Methods and compositions for the repair and/or regeneration of damaged myocardium

ABSTRACT

Methods, compositions, and kits for repairing damaged myocardium and/or myocardial cells including the administration cytokines are disclosed and claimed. Methods and compositions for the development of large arteries and vessels are also disclosed and claimed. The present application also discloses and claims methods and media for the growth, expansion, and activation of human cardiac stem cells.

RELATED APPLICATIONS/PATENT & INCORPORATION BY REFERENCE

This application is a divisional of U.S. patent application Ser. No.11/357,898, now U.S. Pat. No. 7,862,810, filed Feb. 16, 2006, which is acontinuation-in-part of U.S. patent application Ser. No. 10/162,796filed Jun. 5, 2002, now U.S. Pat. No. 7,547,674, which is acontinuation-in-part of U.S. patent application Ser. No. 09/919,732filed Jul. 31, 2001, now abandoned, which claims priority fromProvisional U.S. Patent Application Ser. Nos. 60/295,807, 60/295,806,60/295,805, 60/295,804, and 60/295,803 filed Jun. 6, 2001, 60/258,805,filed Jan. 2, 2001, 60/258,564, filed Dec. 29, 2000, and 60/221,902,filed Jul. 31, 2000. This application is also related to U.S. patentapplication Ser. No. 11/081,884 filed Mar. 16, 2005.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was in part supported by the government, by grants from theNational Institutes of Health, Grant Nos: HL-38132, AG-15756, HL-65577,HL-55757, HL-68088, HL-70897, HL-76794, HL-66923, HL65573, HL-075480,AG-17042 and AG-023071. The government may have certain rights to thisinvention.

Each of the applications and patents cited in this text, including eachof the foregoing cited applications, as well as each document orreference cited in each of the applications and patents (includingduring the prosecution of each issued patent; “application citeddocuments”), and each of the PCT and foreign applications or patentscorresponding to and/or claiming priority from any of these applicationsand patents, and each of the documents cited or referenced in each ofthe application cited documents, are hereby expressly incorporatedherein by reference. More generally, various documents or references arecited in this text, either in a Reference List before the claims or inthe text itself; and, each of the documents or references (“herein citeddocuments”) and all of the documents cited in this text (also “hereincited documents”), as well as each document or reference cited in eachof the herein cited documents (including any manufacturer'sspecifications, instructions, etc. for products mentioned herein and inany document incorporated herein by reference), is hereby expresslyincorporated herein by reference. There is no admission that any of thevarious documents cited in this text are prior art as to the presentinvention. Any document having as an author or inventor person orpersons named as an inventor herein is a document that is not by anotheras to the inventive entity herein. Also, teachings of herein citeddocuments and documents cited in herein cited documents and moregenerally in all documents incorporated herein by reference can beemployed in the practice and utilities of the present invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:PRGT-001_(—)12US_SeqList_ST25.txt, date recorded: Feb. 1, 2011, filesize 2 kilobytes).

FIELD OF THE INVENTION

The present invention relates generally to the field of cardiology, andmore particularly relates to methods and cellular compositions fortreatment of a patient suffering from a cardiovascular disease,including, but not limited to, atherosclerosis, ischemia, hypertension,restenosis, angina pectoris, rheumatic heart disease, congenitalcardiovascular defects and arterial inflammation and other disease ofthe arteries, arterioles and capillaries. The present inventioncontemplates treatments, therapeutics and methodologies that can be usedin place of, or in conjunction with, traditional, invasive therapeutictreatments such as cardiac or vascular bypass surgery.

Moreover, the present invention relates to any one or more of:

Methods and/or pharmaceutical compositions comprising a therapeuticallyeffective amount of somatic stem cells alone or in combination with acytokine such as a cytokine selected from the group consisting of stemcell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), stromalcell-derived factor-1, steel factor, vascular endothelial growth factor,macrophage colony stimulating factor, granulocyte-macrophage stimulatingfactor or Interleukin-3 or any cytokine capable of the stimulatingand/or mobilizing stem cells. Cytokines may be administered alone or incombination of with any other cytokine capable of: the stimulationand/or mobilization of stem cells; the maintenance of early and latehematopoiesis (see below); the activation of monocytes (see below),macrophage/monocyte proliferation; differentiation, motility andsurvival (see below) and a pharmaceutically acceptable carrier, diluentor excipient (including combinations thereof). The stem cells areadvantageously adult stem cells, such as hematopoietic or cardiac stemcells or a combination thereof or a combination of cardiac stem cellsand any other type of stem cells.

The implanting, depositing, administering or causing of implanting ordepositing or administering of stem cells, such as adult stem cells, forinstance hematopoietic or cardiac stem cells or a combination thereof orany combination of cardiac stem cells (e.g., adult cardiac stem cells)and stem cells of another type of (e.g., adult stem cells of anothertype), alone or with a cytokine such as a cytokine selected from thegroup consisting of stem cell factor (SCF), granulocyte-colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), stromal cell-derived factor-1, steel factor, vascularendothelial growth factor, macrophage colony stimulating factor,granulocyte-macrophage stimulating factor or Interleukin-3 or anycytokine capable of the stimulating and/or mobilizing stem cells(wherein “with a cytokine . . . ” can include sequential implanting,depositing administering or causing of implanting or depositing oradministering of the stem cells and the cytokine or the co-implantingco-depositing or co-administering or causing of co-implanting orco-depositing or co-administering or the simultaneous implanting,depositing administering or causing of implanting or depositing oradministering of the stem cells and the cytokine), in circulatory tissueor muscle tissue or circulatory muscle tissue, e.g., cardiac tissue,such as the heart or blood vessels—e.g., veins, arteries, that go to orcome from the heart such as veins and arteries directly connected orattached or flowing into the heart, for instance the aorta. Thisimplanting, depositing, or administering or causing of implanting,depositing or administering can be in conjunction with grafts. Suchimplanting, depositing or administering or causing of implanting,depositing or administering is advantageously employed in the treatmentor therapy or prevention of cardiac conditions, such as to treat areasof weakness or scarring in the heart or prevent the occurrence orfurther occurrence of such areas or to treat conditions which cause orirritate such areas, for instance myocardial infarction or ischemia orother e.g., genetic, conditions that impart weakness or scarring to theheart (see also cardiac conditions mentioned infra).

The use of such stem cells alone or in combination with saidcytokine(s), in the formulation of medicaments for such treatment,therapy or prevention.

Medicaments for use in such treatment, therapy or prevention comprisingthe stem cells and optionally the cytokine(s).

Kits comprising the stem cells and optionally the cytokine(s) forformulations for use in such treatment, therapy or prevention.

Compositions comprising such stem cells and optionally at least onecytokine and kits for preparing such compositions.

Methods of making the kits and compositions described herein.

Methods of implanting or depositing stem cells or causing the implantingor depositing of stem cells.

Methods and/or pharmaceutical compositions comprising a therapeuticallyeffective amount of one or more cytokines for causing the migrationand/or proliferation of cardiac stem cells or cardiac primitive cellsinto circulatory tissue or muscle tissue or circulatory muscle tissue,e.g., cardiac tissue, such as the heart or blood vessels—e.g., veins,arteries, that go to or come from the heart such as veins and arteriesdirectly connected or attached or flowing into the heart, for instancethe aorta. This migration and/or proliferation is advantageouslyemployed in the treatment or therapy or prevention of cardiacconditions, such as to treat areas of weakness or scarring in the heartor prevent the occurrence or further occurrence of such areas or totreat conditions which cause or irritate such areas, for instancemyocardial infarction or ischemia or other e.g., genetic, conditionsthat impart weakness or scarring to the heart (see also cardiacconditions mentioned infra).

Medicaments for use in such treatment, therapy or prevention comprisingthe two or more cytokines.

Kits comprising the cytokines for formulations for use in suchtreatment, therapy or prevention.

Compositions comprising the cytokines and kits for preparing suchcompositions.

Methods of making the kits and compositions described herein.

Methods and/or pharmaceutical compositions comprising a therapeuticallyeffective amount of one or more cytokines for causing the migrationand/or proliferation of cardiac stem cells or cardiac primitive cellsinto circulatory tissue or muscle tissue or circulatory muscle tissue,e.g., cardiac tissue, such as the heart or blood vessels—e.g., veins,arteries, that go to or come from the heart such as veins and arteriesdirectly connected or attached or flowing into the heart, for instancethe aorta in combination with a therapeutically effective amount of apharmaceutical agent useful in treating hypertension, myocardialinfarction, ischemia, angina, or other coronary or vascular ailments,such as AT₁ receptor blockers such as losartan, streptokinase, ReoPro(abciximab), enalapril maleate, Rapilysin (reteplase), Dilatrend(carvedilol), Activase (alteplase), and other drugs for similar useswhich would be known by one skilled in the art.

Methods of treating a patient suffering from hypertension, myocardialinfarction, ischemia, angina or other coronary or vascular ailments,utilizing the above pharmaceutical compositions.

Kits comprising one or more cytokines in combination with apharmaceutical agent useful in treating hypertension, myocardialinfarction, ischemia, angina, or other coronary or vascular ailments.

Methods of making and using the above kits and compositions.

Methods of isolating, expanding and activating stem cells, in particularcardiac stem cells.

Media used in the culture, expansion and/or activation of stem cells, inparticular cardiac stem cells.

Methods of treating occlusions or blockages in arteries and/or vesselscomprising the administration of stem cells, in particular cardiac stemcells, more in particular activated cardiac stem cells, and optionallyin the presence of one or more cytokines.

BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and thereby the principal cause ofdeath in the United States. Atherosclerosis is a complex diseaseinvolving many cell types and molecular factors (for a detailed review,see Ross, 1993, Nature 362: 801-809).

Ischemia is a condition characterized by a lack of oxygen supply intissues of organs due to inadequate perfusion. Such inadequate perfusioncan have number of natural causes, including atherosclerotic orrestenotic lesions, anemia, or stroke, to name a few. Many medicalinterventions, such as the interruption of the flow of blood duringbypass surgery, for example, also lead to ischemia. In addition tosometimes being caused by diseased cardiovascular tissue, ischemia maysometimes affect cardiovascular tissue, such as in ischemic heartdisease. Ischemia may occur in any organ, however, that is suffering alack of oxygen supply.

The most common cause of ischemia in the heart is myocardial infarction(MI), commonly known as a heart attack, is one of the most well-knowntypes of cardiovascular disease. 1998 estimates show 7.3 million peoplein the United States suffer from MI, with over one million experiencingan MI in a given year (American Heart Association, 2000). Of theseindividuals, 25% of men, and 38% of females will die within a year oftheir first recognized MI (American Heart Association, 2000). MI iscaused by a sudden and sustained lack of blood flow to an area of theheart, commonly caused by narrowing of a coronary artery. Withoutadequate blood supply, the tissue becomes ischemic, leading to the deathof myocytes and vascular structures. This area of necrotic tissue isreferred to as the infarct site, and will eventually become scar tissue.Survival is dependent on the size of this infarct site, with theprobability of recovery decreasing with increasing infarct size. Forexample, in humans, an infarct of 46% or more of the left ventricletriggers irreversible cardiogenic shock and death (99).

Current treatments for MI focus on reperfusion therapy, which attemptsto start the flow of blood to the affected area to prevent the furtherloss of tissue. The main choices for reperfusion therapy include the useof anti-thrombolytic agents, or performing balloon angioplasty, or acoronary artery bypass graft. Anti-thrombolytic agents solubilize bloodclots that may be blocking the artery, while balloon angioplasty threadsa catheter into the artery to the site of the occlusion, where the tipof the catheter is inflated, pushing open the artery. Still moreinvasive procedures include the bypass, where surgeons remove a sectionof a vein from the patient, and use it to create a new artery in theheart, which bypasses the blockage, and continues the supply of blood tothe affected area. In 1998, there were an estimated 553,000 coronaryartery bypass graft surgeries and 539,000 percutaneous transluminalcoronary angioplastys. These procedures average $27,091 and $8,982 perpatient, respectively (American Heart Association, 2000).

These treatments may succeed in reestablishing the blood supply, howevertissue damage that occurred before the reperfusion treatment began hasbeen thought to be irreversible. For this reason, eligible MI patientsare started on reperfusion therapy as soon as possible to limit the areaof the infarct.

As such, most studies on MI have also focused on reducing infarct size.There have been a few attempts to regenerate the necrotic tissue bytransplanting cardiomyocytes or skeletal myoblasts (Leor et al., 1996;Murray, et al., 1996; Taylor, et al., 1998; Tomita et al., 1999;Menasche et al., 2000). While the cells may survive aftertransplantation, they fail to reconstitute healthy myocardium andcoronary vessels that are both functionally and structurally sound.

All of the cells in the normal adult originate as precursor cells whichreside in various sections of the body. These cells, in turn, derivefrom very immature cells, called progenitors, which are assayed by theirdevelopment into contiguous colonies of cells in 1-3 week cultures insemisolid media such as methylcellulose or agar. Progenitor cellsthemselves derive from a class of progenitor cells called stem cells.Stem cells have the capacity, upon division, for both self-renewal anddifferentiation into progenitors. Thus, dividing stem cells generateboth additional primitive stem cells and somewhat more differentiatedprogenitor cells. In addition to the well-known role of stem cells inthe development of blood cells, stem cells also give rise to cells foundin other tissues, including but not limited to the liver, brain, andheart.

Stem cells have the ability to divide indefinitely, and to specializeinto specific types of cells. Totipotent stem cells, which exist afteran egg is fertilized and begins dividing, have total potential, and areable to become any type of cell. Once the cells have reached theblastula stage, the potential of the cells has lessened, with the cellsstill able to develop into any cell within the body, however they areunable to develop into the support tissues needed for development of anembryo. The cells are considered pluripotent, as they may still developinto many types of cells. During development, these cells become morespecialized, committing to give rise to cells with a specific function.These cells, considered multipotent, are found in human adults andreferred to as adult stem cells. It is well known that stem cells arelocated in the bone marrow, and that there is a small amount ofperipheral blood stem cells that circulate throughout the blood stream(National Institutes of Health, 2000).

Due to the regenerative properties of stem cells, they have beenconsidered an untapped resource for potential engineering of tissues andorgans. It would be an advance to provide uses of stem cells withrespect to addressing cardiac conditions.

Mention is made of:

U.S. Pat. No. 6,117,675 which relates to the differentiation of retinalstem cells into retinal cells in vivo or in vitro, which can be used asa therapy to restore vision.

U.S. Pat. No. 6,001,934 involving the development of functional isletsfrom islets of Langerhans stem cells.

U.S. Pat. Nos. 5,906,934 and 6,174,333 pertaining to the use ofmesenchymal stem cells for cartilage repair, and the use of mesenchymalstem cells for regeneration of ligaments; for instance, wherein the stemcells are embedded in a gel matrix, which is contracted and thenimplanted to replace the desired soft tissue.

U.S. Pat. Nos. 6,099,832, and 6,110,459 involving grafts with celltransplantation.

PCT Application Nos. PCT/US00/08353 (WO 00/57922) and PCT/US99/17326 WO00/06701) involving intramyocardial injection of autologous bone marrowand mesenchymal stem cells which fails to teach or suggestadministering, implanting, depositing or the use of hematopoietic stemcells as in the present invention, especially as hematopoietic stemcells as in the present invention are advantageously isolated and/orpurified adult hematopoietic stem cells.

Furthermore, at least certain of these patent documents fail to teach orsuggest the present invention for additional reasons. The source of thestem cells of interest is limited to the known precursors of the type oftissue for which regeneration is required. Obtaining and purifying thesespecific cells can be extremely difficult, as there are often very fewstem cells in a given tissue. In contrast, a benefit of the presentinvention results from the ability of various lineages of stem cells tohome to the myocardium damage and differentiate into the appropriatecell types—an approach that does not require that the stem cells arerecovered directly from myocardium, and, a variety of types of stemcells may be used without compromising the functionality of theregenerated tissue. And, other of these patent documents utilize stemcells as the source of various chemical compositions, without utilizingtheir proliferative capabilities, and thereby fail to teach or suggestthe invention.

Only recent literature has started to investigate the potentials forstem cells to aid in the repair of tissues other than that of knownspecialization. This plasticity of stem cells, the ability to cross theborder of germ layers, is a concept only in its infancy (Kempermann etal, 2000, Temple, 2001). Kocher et al (2001) discusses the use of adultbone marrow to induce neovascularization after infarction as analternative therapy for left ventricle remodeling (reviewed in Rosenthaland Tsao, 2001). Other studies have focused on coaxing specific types ofstem cells to differentiate into myocardial cells, i.e. liver stem cellsas shown in Malour et al (2001). Still other work focuses on thepossibilities of bone-marrow derived stem cells (Krause, et al., 2001).

One of the oldest uses of stem cells in medicine is for the treatment ofcancer. In these treatments, bone marrow is transplanted into a patientwhose own marrow has been destroyed by radiation, allowing the stemcells in the transplanted bone marrow to produce new, healthy, whiteblood cells.

In these treatments, the stem cells are transplanted into their normalenvironment, where they continue to function as normal. Until recently,it was thought that any particular stem cell line was only capable ofproducing three or four types of cells, and as such, they were onlyutilized in treatments where the stem cell was required to become one ofthe types of cells for which their ability was already proven.Researchers are beginning to explore other options for treatments ofmyriad disorders, where the role of the stem cell is not well defined.Examples of such work will be presented in support of the presentinvention.

Organ transplantation has been widely used to replace diseased,nonfunctional tissue. More recently, cellular transplantation to augmentdeficiencies in host tissue function has emerged as a potentialtherapeutic paradigm. One example of this approach is the wellpublicized use of fetal tissue in individuals with Parkinsonism(reviewed in Tompson, 1992), where dopamine secretion from transplantedcells alleviates the deficiency in patients. In other studies,transplanted myoblasts from unaffected siblings fused with endogenousmyotubes in Duchenne's patients; importantly the grafted myotubesexpressed wild-type dystrophin (Gussoni et al., 1992).

Despite their relevance in other areas, these earlier studies do notdescribe any cellular transplantation technology that can besuccessfully applied to the heart, where the ability to replace damagedmyocardium would have obvious clinical relevance. Additionally, the useof intra-cardiac grafts to target the long-term expression of angiogenicfactors and ionotropic peptides would be of therapeutic value forindividuals with myocardial ischemia or congestive heart failure,respectively.

In light of this background there is a need for the improvement ofmyocardial regeneration technology in the heart. Desirably, suchtechnology would not only result in tissue regeneration in the heart butalso enable the delivery of useful compositions directly to the heart.The present invention addresses these needs.

It is therefore believed that heretofore the administration, implanting,depositing, causing to be deposited, implanted or administered of stemcells, alone or in combination with at least one cytokine, as well asthe use of such stem cells alone or in combination with saidcytokine(s), in the formulation of medicaments for treatment, therapy orprevention, as in this disclosure and as in the present invention, hasnot been taught, or suggested in the art and that herein methods,compositions, kits and uses are novel, nonobvious and inventive, i.e.,that the present invention has not been taught or suggested in the artand that the present invention is novel, nonobvious and inventive.

OBJECT AND SUMMARY OF THE INVENTION

It has surprisingly been found that the implantation of somatic stemcells into the myocardium surrounding an infarct following a myocardialinfarction, migrate into the damaged area, where they differentiate intomyocytes, endothelial cells and smooth muscle cells and then proliferateand form structures including myocardium, coronary arteries, arterioles,and capillaries, restoring the structural and functional integrity ofthe infarct.

It has also surprisingly been found that following a myocardialinfarction, the administration of a cytokine to the patient, stimulatesthe patient's own resident and/or circulating stem cells, causing themto enter the blood stream and home to the infarcted area. It has alsobeen found that once the cells home to the infarct, they migrate intothe damaged tissue, where they differentiate into myocytes, endothelialcells and smooth muscle cells and then proliferate and form structuresincluding myocardium, coronary arteries, arterioles and capillaries,restoring structural and functional integrity to the infracted area.

Surprisingly, resident cardiac stem cells (CSCs) have recently beenidentified in the human (82) and rat (83, 84) heart. These primitivecells tend to accumulate in the atria (82) although they are alsopresent throughout the ventricular myocardium (82, 83, 84). CSCs expresssurface antigens commonly found in hematopoietic and skeletal musclestem cells (85, 86). CSCs are clonogenic, self-renewing and multipotentgiving rise to all cardiac lineages (84). Because of the growthproperties of CSCs, the injured heart has the potential to repairitself. However, this possibility had been limited by our lack ofunderstanding of CSC colonization, proliferation and differentiation innew organized, functioning myocardium (61, 87). Identical obstaclesapply to any other source of stem cells in the organism (88).

The identification of c-Met on hematopoietic and hepatic stem cells (89,90, 91) and, most importantly, on satellite skeletal muscle cells (92)has prompted the determining of whether its ligand, hepatocyte growthfactor (HGF), has a biological effect on CSCs. Assuming that HGFmobilize and promote the translocation of CSCs from anatomical storageareas to the site of damage acutely after infarction. HGF positivelyinfluences cell migration (93) through the expression and activation ofmatrix metalloproteinase-2 (94, 95). This enzyme family destroysbarriers in the extracellular matrix thereby facilitating CSC movement,homing and tissue restoration.

Similarly, insulin-like growth factor-1 (IGF-1) is mitogenic,antiapoptotic and is necessary for neural stem cell multiplication anddifferentiation (96, 97, 98). In a comparable manner, IGF-1 impacts CSCsby increasing their number and protecting their viability. IGF-1overexpression is characterized by myocyte proliferation in the adultmouse heart (65) and this cell growth may depend on CSC activation,differentiation and survival.

Consequently, the invention provides methods and/or compositions forrepairing and/or regenerating recently damaged myocardium and/ormyocardial cells comprising the administration of an effective amount ofone or more cytokines, e.g. HGF and IGF-1 for causing the migrationand/or proliferation of cardiac stem cells or cardiac primitive cellsinto circulatory tissue or muscle tissue or circulatory muscle tissue.This migration and/or proliferation is advantageously employed in thetreatment or therapy or prevention of cardiac conditions, such as totreat areas of weakness or scarring in the heart or prevent theoccurrence or further occurrence of such areas or to treat conditionswhich cause or irritate such areas, for instance myocardial infarctionor ischemia or other, e.g. genetic, conditions that impart weakness orscarring to the heart.

It is reasonable to suggest that the protocol used here is superior tothe procedure employed to replace the necrotic or scarred myocardium bytransplanting cardiomyocytes (42, 79), skeletal myoblasts (55, 76) orthe prospective utilization of embryonic cells (100, 101). Althoughthese attempts have been successful in the survival of many of thegrafted cells, they have failed to reconstitute healthy myocardium andcoronary vessels integrated structurally and functionally with thespared portion of the ventricular wall. CSCs are programmed to regulatethe normal cell turnover of the heart and, under stressful conditions,participate in the recovery of the injured ventricle structurally andmechanically (82, 102).

The invention also provides methods and/or compositions comprising atherapeutically effective amount of one or more cytokines for causingthe migration and/or proliferation of cardiac stem cells or cardiacprimitive cells into circulatory tissue or muscle tissue or circulatorymuscle tissue. This migration and/or proliferation is advantageouslyemployed in the treatment or therapy or prevention of cardiacconditions, such as to treat areas of weakness or scarring in the heartor prevent the occurrence or further occurrence of such areas or totreat conditions which cause or irritate such areas, for instancemyocardial infarction or ischemia or other, e.g. genetic, conditionsthat impart weakness or scarring to the heart.

The invention also provides medicaments for use in such treatment,therapy or prevention.

The invention further provides kits comprising one or more cytokines forformulation for use in such treatment, therapy or prevention.

The invention still further provides methods of making the kits andcompositions described herein.

The invention further provides compositions and/or kits comprising oneor more cytokines in combination with a therapeutic agent for treatingcardiac or vascular conditions for formulation for use in suchtreatment, therapy or prevention.

The invention provides to methods and/or compositions for repairingand/or regenerating recently damaged myocardium and/or myocardial cellscomprising the administration of somatic stem cells, e.g., adult stemcells or cardiac stem cells or hematopoietic stem cells or a combinationthereof, such as adult cardiac or adult hematopoietic stem cells or acombination thereof or a combination of cardiac stem cells and a stemcell of another type, such as a combination of adult cardiac stem cellsand adult stem cells of another type.

In one aspect, the invention provides media for use in the culturingand/or expansion of stem cells in vitro, prior to the administration ofthe stem cells.

The invention further provides a method and/or compositions forrepairing and/or regenerating recently damaged myocardium and/ormyocardial cells comprising the administration of at least one cytokine.

The invention further provides methods and/or compositions for repairingand/or regenerating recently damaged myocardium and/or myocardial cellscomprising the administration of at least one cytokine in combinationwith a pharmaceutical agent useful in the treatment of cardiac orvascular conditions.

The invention still further relates to a method and/or compositions forrepairing and/or regenerating recently damaged myocardium comprising theadministration of somatic stem cells, e.g., adult stem cells or cardiacstem cells or hematopoietic stem cells or a combination thereof, such asadult cardiac or adult hematopoietic stem cells or a combination thereofor a combination of cardiac stem cells and a stem cell of another type,such as a combination of adult cardiac stem cells and adult stem cellsof another type and a cytokine.

The invention yet further provides a method for preparing any of theaforementioned or herein disclosed compositions comprising admixing thepharmaceutically acceptable carrier and the somatic stem cells and/orcytokines.

The invention also provides to a kit comprising a pharmaceuticalcomposition for use in repairing and/or regenerating recently damagedmyocardium and/or myocardial cells.

The invention provides methods involving implanting, depositing,administering or causing the implanting or depositing or administeringof stem cells, such as adult stem cells, for instance hematopoietic orcardiac stem cells or a combination thereof or any combination ofcardiac stem cells (e.g., adult cardiac stem cells) and stem cells ofanother type of (e.g., adult stem cells of another type), alone or witha cytokine such as a cytokine selected from the group consisting of stemcell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), stromalcell-derived factor-1, steel factor, vascular endothelial growth factor,macrophage colony stimulating factor, granulocyte-macrophage stimulatingfactor or interleukin-3 or any cytokine capable of the stimulatingand/or mobilizing stem cells (wherein “with a cytokine . . . ” caninclude sequential implanting, depositing administering or causing ofimplanting or depositing or administering of the stem cells and thecytokine or the co-implanting co-depositing or co-administering orcausing of co-implanting or co-depositing or co-administering or thesimultaneous implanting, depositing administering or causing ofimplanting or depositing or administering of the stem cells and thecytokine), in circulatory tissue or muscle tissue or circulatory muscletissue, e.g., cardiac tissue, such as the heart or blood vessels—e.g.,veins, arteries, that go to or come from the heart such as veins andarteries directly connected or attached or flowing into the heart, forinstance the aorta. This implanting, depositing, or administering orcausing of implanting, depositing or administering can be in conjunctionwith grafts.

Such implanting, depositing or administering or causing of implanting,depositing or administering is advantageously employed in the treatmentor therapy or prevention of cardiac conditions, such as to treat areasof weakness or scarring in the heart or prevent the occurrence orfurther occurrence of such areas or to treat conditions which cause orirritate such areas, for instance myocardial infarction or ischemia orother e.g., genetic, conditions that impart weakness or scarring to theheart (see also cardiac conditions mentioned supra).

The invention additionally provides the use of such stem cells alone orin combination with said cytokine(s), in the formulation of medicamentsfor such treatment, therapy or prevention.

And thus, the invention also provides medicaments for use in suchtreatment, therapy or prevention comprising the stem cells andoptionally the cytokine(s).

Likewise the invention provides kits comprising the stem cells andoptionally the cytokine(s) for formulations for use in such treatment,therapy or prevention. The stem cells and the cytokine(s) can be inseparate containers in a package or in one container in a package; and,the kit can optionally include a device for administration (e.g.,syringe) and/or instructions for administration and/or admixture.

The invention also provides compositions comprising such stem cells andoptionally the cytokine(s) and kits for preparing such compositions(e.g., kits comprising the stem cells and optionally the cytokine(s);stem cells and the cytokine(s) can be in separate containers in apackage or in one container in a package; and, the kit can optionallyinclude a device for administration (e.g., syringe) and/or instructionsfor administration and/or admixture), as well as methods of making theaforementioned compositions.

The invention also provides a means of generating and/or regeneratingmyocardium ex vivo, wherein somatic stem cells and heart tissue arecultured in vitro, optionally in the presence of a cytokine. The somaticstem cells differentiate into myocytes, smooth muscle cells andendothelial cells, and proliferate in vitro, forming myocardial tissueand/or cells. These tissues and cells may assemble into cardiacstructures including arteries, arterioles, capillaries, and myocardium.The tissue and/or cells formed in vitro may then be implanted into apatient, e.g. via a graft, to restore structural and functionalintegrity.

The invention additionally provides a means of generating large vesselsuseful in the treatment of occlusion or blockage of an artery or vessel.Such methods can be used in place or, or in conjunction with,traditional methods of cardiac bypass surgery. The methods of theinvention herein relate to the isolation, expansion, and activation ofcardiac stem cells, wherein the cardiac stem cells are activated throughcontact with one or more stem cells. The activated cardiac stem cellsare then delivered or implanted at the site of the blockage orocclusion.

Furthermore, the invention provides growth media that can be used theculture and expansion of stem cells, in particular cardiac stem cells.Also provided is growth media that can be used to activate stem cells,in particular cardiac stem cells. Non-activated stem cells grown in saidmedia can be administered to regenerate myocardium or vasculature.Activated stem cells grown in the media can also be administered toregenerate myocardium or vasculature, wherein vasculature includes largearteries and veins, such as in a biological bypass.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The methods of the present invention are considered biotechnologymethods in under 35 U.S.C. §287(c)(2)(A)(iii) which provides that theinfringement exception for medical activity does not apply to thepractice of a process in violation of a biotechnology patent.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF FIGURES

The following Detailed Description, given to describe the invention byway of example, but not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figures, incorporated herein by reference, in which:

FIG. 1 shows a log-log plot showing Lin⁻ bone marrow cells from EGFPtransgenic mice sorted by FACS based on c-kit expression (The fractionof c-kit^(POS) cells (upper gate) was 6.4%. c-kit^(NEG) cells are shownin the lower gate. c-kit^(POS) cells were 1-2 logs brighter thanc-kit^(NEG) cells)

FIG. 2A shows a photograph of a tissue section from a MI induced mouse(The photograph shows the area of myocardial infarct (MI) injected withLin⁻ c-kit^(POS) cells from bone marrow (arrows), the remaining viablemyocardium (VM), and the regenerating myocardium (arrowheads).Magnification is 12×);

FIG. 2B shows a photograph of the same tissue section of FIG. 2A at ahigher magnification, centering on the area of the MI with magnificationbeing 50×;

FIG. 2C, D show photographs of a tissue section at low and highmagnifications of the area of MI, injected with Lin⁻ c-kit^(POS) cells,with the magnification of 2C being 25×, and the magnification of 2Dbeing 50×;

FIG. 2E shows a photograph of a tissue section of the area of MIinjected with Lin⁻ c-kit^(NEG) cells wherein only healing is apparentand the magnification is 50× (*Necrotic myocytes. Red=cardiac myosin;green=PI labeling of nuclei);

FIG. 3A-C show photographs of a section of tissue from a MI inducedmouse, showing the area of MI injected with Lin⁻ c-kit^(POS) cells(Visible is a section of regenerating myocardium from endocardium (EN)to epicardium (EP). All photographs are labeled to show the presence ofinfarcted tissue in the subendocardium (IT) and spared myocytes in thesubendocardium (SM). FIG. 3A is stained to show the presence of EGFP(green). Magnification is 250×. FIG. 3B is stained to show the presenceof cardiac myosin (red). Magnification is 250×. FIG. 3C is stained toshow the presence of both EGFP and myosin (red-green), as well asPI-stained nuclei (blue). Magnification is 250×);

FIG. 4A shows of grafts depicting the effects of myocardial infarctionon left ventricular end-diastolic pressure (LVEDP), developed pressure(LVDP), LV+ rate of pressure rise (dP/dt), and LV-rate of pressure decay(dP/dt) (From left to right, bars indicate: sham-operated mice (SO,n=11); mice non-injected with Lin⁻ c-kit^(POS) cells (MI, n=5 injectedwith Lin⁻ c-kit^(NEG) cells; n=6 non-injected); mice injected with Lin⁻c-kit^(POS) cells (MI+BM, n=9). Error bars are the standard deviation.*, † p<0.05 vs SO and MI);

FIG. 4B shows a drawing of a proposed scheme for Lin⁻ c-kit^(POS) celldifferentiation in cardiac muscle and functional implications;

FIG. 5A-I show photographs of a tissue sections from a MI induced mousedepicting regenerating myocardium in the area of the MI which has beeninjected with Lin⁻ c-kit^(POS) cells (FIG. 5A is stained to show thepresence of EGFP (green). Magnification is 300×. FIG. 5B is stained toshow the presence of α-smooth muscle actin in arterioles (red).Magnification is 300×. FIG. 5C is stained to show the presence of bothEGFP and α-smooth muscle actin (yellow-red), as well as PI-stainednuclei (blue). Magnification is 300×. FIGS. 5D-F and G-I depict thepresence of MEF2 and Csx/NRx2.5 in cardiac myosin positive cells. FIG.5D shows PI-stained nuclei (blue). Magnification is 300×. FIG. 5E isstained to show MEF2 and Csx/NRx2.5 labeling (green). Magnification is300×. FIG. 5F is stained to show cardiac myosin (red), as well as MEF2or Csx/NRx2.5 with PI (bright fluorescence in nuclei). Magnification is300×. FIG. 5G shows PI-stained nuclei (blue). Magnification is 300×.FIG. 5H is stained to show MEF2 and Csx/NRx2.5 labeling (green).Magnification is 300×. FIG. 5I is stained to show cardiac myosin (red),as well as MEF2 or Csx/NRx2.5 with PI (bright fluorescence in nuclei).Magnification is 300×);

FIG. 6 (FIG. 6A-F) shows photographs of tissue sections from MI inducedmice, showing regenerating myocardium in the area of the MI injectedwith Lin⁻ c-kit^(POS) cells (FIG. 6A-C show tissue which has beenincubated in the presence of antibodies to BrdU. FIG. 6A has beenstained to show PI-labeled nuclei (blue). Magnification is 900×. FIG. 6Bhas been stained to show BrdU- and Ki67-labeled nuclei (green).Magnification is 900×. FIG. 6C has been stained to show the presence ofα-sarcomeric actin (red). Magnification is 900×. FIG. 6D-F shows tissuethat has been incubated in the presence of antibodies to Ki67. FIG. 6Dhas been stained to show PI-labeled nuclei (blue). Magnification is500×. FIG. 6E has been stained to show BrdU- and Ki67-labeled nuclei(green). Magnification is 500×. FIG. 6F has been stained to show thepresence of α-smooth muscle actin (red). Magnification is 500×. Brightfluorescence: combination of PI with BrdU (C) or Ki67 (F));

FIG. 7 (FIG. 7A-C) shows photographs of tissue sections from MI inducedmice, showing the area of MI injected with Lin⁻ c-kit^(POS) cells(Depicted are the border zone, viable myocardium (VM) and the new band(NB) of myocardium separated by an area of infarcted non-repairingtissue (arrows). FIG. 7A is stained to show the presence of EGFP(green). Magnification is 280×. FIG. 7B is stained to show the presenceof cardiac myosin (red). Magnification is 280×. FIG. 7C is stained toshow the presence of both EGFP and myosin (red-green), as well asPI-stained nuclei (blue). Magnification is 280×);

FIG. 8 (FIG. 8A-F) shows photographs of tissue sections from MI inducedmice, showing regenerating myocardium in the area of MI injected withLin⁻ c-kit^(POS) cells (FIG. 8A is stained to show the presence of EGFP(green). Magnification is 650×. FIG. 8B is stained to show the presenceof cardiac myosin (red). Magnification is 650×. FIG. 8C is stained toshow both the presence of EGFP and myosin (yellow), as well asPI-stained nuclei (blue). Magnification is 650×. FIG. 8D is stained toshow the presence of EGFP (green). Magnification is 650×. FIG. 8E isstained to show the presence of α-smooth muscle actin in arterioles(red). Magnification is 650×. FIG. 8F is stained to show the presence ofboth EGFP and α-smooth muscle actin (yellow-red) as well as PI-stainednuclei (blue). Magnification is 650×);

FIG. 9 (FIG. 9A-C) shows photographs of tissue sections from MI inducedmice, showing the area of MI injected with Lin⁻ c-kit^(POS) cells andshowing regenerating myocardium (arrowheads). (FIG. 9A is stained toshow the presence of cardiac myosin (red) Magnification is 400×. FIG. 9Bis stained to show the presence of the Y chromosome (green).Magnification is 400×. FIG. 9C is stained to show both the presence ofthe Y chromosome (light blue) and PI-labeled nuclei (dark blue). Notethe lack of Y chromosome in infarcted tissue (IT) in subendocardium andspared myocytes (SM) in subepicardium. Magnification is 400×);

FIG. 10 (FIG. 10A-C) shows photographs of tissue sections from MIinduced mice, showing GATA-4 in cardiac myosin positive cells (FIG. 10Ashows PI-stained nuclei (blue). Magnification is 650×. FIG. 10B showsthe presence of GATA-4 labeling (green). Magnification is 650×. FIG. 10Cis stained to show cardiac myosin (red) in combination with GATA-4 andPI (bright fluorescence in nuclei). Magnification is 650×);

FIG. 11 (FIG. 11A-D) shows photograph of tissue sections from a MIinduced mouse (FIG. 11A shows the border zone between the infarctedtissue and the surviving tissue. Magnification is 500×. FIG. 11B showsregenerating myocardium. Magnification is 800×. FIG. 11C is stained toshow the presence of connexin 43 (yellow-green), and the contactsbetween myocytes are shown by arrows. Magnification is 800×. FIG. 11D isstained to show both α-sarcomeric actin (red) and PI-stained nuclei(blue). Magnification is 800×);

FIG. 12 (FIG. 12A-B) shows photographs of tissue sections from a MIinduced mouse showing the area of MI that was injected with Lin⁻c-kit^(POS) cells and now shows regenerating myocytes (FIG. 12A isstained to show the presence of cardiac myosin (red) and PI-labelednuclei (yellow-green). Magnification is 1,000. FIG. 12B is the same asFIG. 12A at a magnification of 700×);

FIG. 13A-B show photographs of tissue sections from MI induced mice(FIG. 13A shows a large infarct (MI) in a cytokine-treated mouse withforming myocardium (arrowheads) (Magnification is 50×) at highermagnification (80×-adjacent panel). FIG. 13B shows a MI in a non-treatedmouse. Healing comprises the entire infarct (arrowheads) (Magnificationis 50×). Scarring is seen at higher magnification (80×-adjacent panel).Red=cardiac myosin; yellow-green=propidium iodide (PI) labeling ofnuclei; blue-magenta=collagen types I and III);

FIG. 13C shows a graph showing the mortality and myocardial regenerationin treated and untreated MI induced mice (Cytokine-treated infarctedmice, n=15; untreated infarcted mice, n=52. Log-rank test: p<0.0001);

FIG. 14 shows a graph showing quantitative measurement of infarct size(Total number of myocytes in the left ventricular free wall (LVFW) ofsham-operated (SO, n=9), infarcted non-treated (MI, n=9) andcytokine-treated (MI-C, n=11) mice at sacrifice, 27 days afterinfarction or sham operation. The percentage of myocytes lost equalsinfarct size. X±SD, *p<0.05 vs SO);

FIG. 15A-C show graphs comparing aspects of myocardial infarction,cardiac anatomy and function (FIG. 15A-C depict LV dimensions atsacrifice, 27 days after surgery; sham-operated (SO, n=9), non-treatedinfarcted (MI, n=9) and cytokine-treated infarcted (MI-C, n=10));

FIG. 15D shows EF by echocardiography; (SO, n=9; MI, n=9; and MI-C,n=9);

FIG. 15E-M show M-mode echocardiograms of SO (e-g), MI (h-j) and MI-C(k-m) (Newly formed contracting myocardium (arrows));

FIG. 15N shows a graph showing wall stress; SO (n=9), MI (n=8) and MI-C(n=9) (Results are mean±SD. *,**p<0.05 vs SO and MI, respectively);

FIG. 16A-G show grafts depicting aspects of myocardial infarction,cardiac anatomy and ventricular function (FIG. 16A-D showechocardiographic LVESD (a), LVEDD (b), PWST (c) and PWDT (d) in SO(n=9), MI (n=9) and MI-C (n=9). FIG. 16E-G show mural thickness (e),chamber diameter (f) and longitudinal axis (g) measured anatomically atsacrifice in SO (n=9), MI (n=9) and MI-C (n=10). ***p<0.05 vs SO and MI,respectively;

FIG. 16H-P show two dimensional (2D) images and M-mode tracings of SO(h-j), MI (k-m) and MI-C (n-p);

FIG. 17 (FIG. 17A-D) shows graphs depicting aspects of ventricularfunction (FIG. 17A-D show LV hemodynamics in anesthetized mice atsacrifice, 27 days after infarction or sham operation; SO (n=9), MI(n=9) and MI-C (n=10). For symbols and statistics, see also FIG. 13);

FIG. 18A-E shows graphs of aspects of myocardial regeneration (FIG. 18Aclassifies the cells in the tissue as remaining viable (Re), lost (Lo)and newly formed (Fo) myocardium in LVFW at 27 days in MI and MI-C; SO,myocardium without infarct. FIG. 18B shows the amount of cellularhypertrophy in spared myocardium. FIG. 18C shows cell proliferation inthe regenerating myocardium. Myocytes (M), EC and SMC labeled by BrdUand Ki67; n=11. *,**p<0.05 vs M and EC. FIG. 18D-E depict the volume,number (n=11) and class distribution (bucket size, 100 μm³; n=4,400) ofmyocytes within the formed myocardium;

FIG. 18F-H show photographs of tissue sections from MI induced micedepicting arterioles with TER-119 labeled erythrocyte membrane (greenfluorescence); blue fluorescence=PI staining of nuclei; redfluorescence=α-smooth muscle actin in SMC (FIG. 18F is magnified at800×. FIG. 18G-H are magnified at 1,200×);

FIG. 19 (FIG. 19A-D) shows photographs of tissue sections from MIinduced mice that were incubated with antibodies to Ki67 (A,B) and BrdU(C,D) (FIG. 19A shows labeling of myocytes by cardiac myosin. Brightfluorescence of nuclei reflects the combination of PI and Ki67.Magnification is 800×. FIG. 19B shows labeling of SMC by α-smooth muscleactin. Bright fluorescence of nuclei reflects the combination of PI andKi67. Magnification is 1,200×.

FIG. 19C shows labeling of SMC by α-smooth muscle actin. Brightfluorescence of nuclei reflects the combination of PI and BrdU.Magnification is 1,200×. FIG. 19D shows labeling of EC in the formingmyocardium by factor VIII. Bright fluorescence of nuclei reflects thecombination of PI and BrdU. Magnification is 1,600×;

FIG. 20 (FIG. 20A-F) shows photographs of tissue sections from MIinduced mice showing markers of differentiating cardiac cells (FIG. 20Ais stained to show labeling of myocytes by nestin (yellow)). Redfluorescence indicates cardiac myosin. Magnification is 1,200×. FIG. 20Bis stained to show labeling of desmin (red). Magnification is 800×. FIG.20C is stained to show labeling of connexin 43 (green). Red fluorescenceindicates cardiac myosin. Magnification is 1,400×. FIG. 20D showsVE-cadherin and yellow-green fluorescence reflects labeling of EC byflk-1 (arrows). Magnification is 1,800×. FIG. 20E shows red fluorescenceindicating factor VIII in EC and yellow-green fluorescence reflectslabeling of EC by flk-1 (arrows). Magnification is 1,200×. FIG. 20Fshows green fluorescence labeling of SMC cytoplasms by flk-1 andendothelial lining labeled by flk-1. Red fluorescence indicates α-smoothmuscle actin. Blue fluorescence indicates PI labeling of nuclei.Magnification is 800×; and

FIG. 21A-C show tissue sections from MI induced mice (FIG. 21A usesbright fluorescence to depict the combination of PI labeling of nucleiwith Csx/NRx2.5. Magnification is 1,400×. FIG. 21B uses brightfluorescence to depict the combination of PI labeling of nuclei withGATA-4. Magnification is 1,200×. FIG. 21C uses bright fluorescence todepict the combination of PI labeling of nuclei with MEF2. Magnificationis 1,200× (Red fluorescence shows cardiac myosin antibody staining andblue fluorescence depicts PI labeling of nuclei. The fraction of myocytenuclei labeled by Csx/NRx2.5, GATA-4 and MEF2 was 63±5% (nucleisampled=2,790; n=11), 94±9% (nuclei sampled=2,810; n=11) and 85±14%(nuclei sampled=3,090; n=11), respectively).

FIG. 22A-L are confocal micrographs which show cardiac primitive cellsin normal and growth factor-treated and untreated infarcted hearts. FIG.22A-F shows sections of atrial myocardium from sham-operated mice. FIGS.22A and B, 22C and D, and 22E and F are pairs of micrographs showing thesame area of atrial myocardium with different stains. c-Met (22A,yellow) is expressed in c-kit^(POS) (22B, green) cells (22B,yellow-green). Similarly, IGF-1R (22C, yellow) is detected in MDR1^(POS)(22D, green) cells (22D, yellow-green). Colocalization of c-Met (22E,red) and IGF-1R (22E, yellow) are found in MDR1^(POS) (22F, green) cells(22F, red-yellow-green). Arrows point to c-Met and IGF-1R in c-kit^(POS)and MDR1^(POS) cells. Myocyte cytoplasm is stained red-purple andcontains cardiac myosin. FIG. 22G: The yellow line separates theinfarcted myocardium (MI) with apoptotic myocytes (bright nuclei, PI andhairpin 1) from the border zone (BZ) with viable myocytes (blue nuclei,PI only) in a mouse treated with growth factors. Viable c-kit^(POS)cells (blue nuclei, PI; c-kit, green) are present in MI and BZ (arrows).Myocyte cytoplasm is stained red and contains cardiac myosin. FIG. 22H:The yellow line separates the MI with necrotic myocytes (bright nuclei,PI and hairpin 2) from the BZ with viable myocytes (blue nuclei, PIonly) in a mouse treated with growth factors. Viable MDR1^(POS) cells(blue nuclei, PI; MDR1, green) are present in MI and BZ (arrows).Myocyte cytoplasm is stained red and contains cardiac myosin). FIGS. 22Iand 22J: Apoptotic myocytes (22I and 22J, bright nuclei, PI andhairpin 1) and c-kit^(POS) (22I, green ring) and MDR1^(POS) (22J, greenring) cells undergo apoptosis (22I and 22J, bright nuclei, PI andhairpin 1; arrows) in the infarcted region of two untreated mice. Viablecells have blue nuclei (PI only). A viable c-kit^(POS) cell is presentwithin the infarcted myocardium (22I, green ring, blue nucleus, PI only;arrowhead). Myocyte cytoplasm is stained red and shows cardiac myosin.FIGS. 22K and 22L: Cycling c-kit^(POS) (22K, green ring; arrows) andMDR1^(POS) (22L, green ring; arrows) cells are present in the infarctedmyocardium (yellow dots are apoptotic nuclei) of mice treated withgrowth factors. Bright fluorescence in c-kit^(POS) (22K) and MDR1^(POS)(22L) cells corresponds to Ki67 labeling of their nuclei. FIGS. 22A-L,bar=10 μm. FIGS. 22M and 22N are graphs depicting the distribution ofviable and dead c-kit^(POS) (22M) and MDR1^(POS) (22N) cells in thevarious regions of the heart in sham-operated (SO), infarcted-treated(Treated) and infarcted-untreated (Untreated) mice sacrificed 7-8 hoursafter surgery and 2-3 hours after the administration of growth factors(Treated) or saline (SO; Untreated). Abbreviations are as follows: A,atria; LV, left ventricle; R, viable myocardium remote from the infarct;B, viable myocardium bordering the infarct; I, non-viable infarctedmyocardium. Results in both FIGS. 22M and 22N are presented as themean±SD. *,** Indicates P<0.05 vs. SO and vs. Treated, respectively.

FIG. 23A-B are graphs depicting the size of infarct and the evaluationof left ventricle hemodynamics. Results are presented as the mean±SD.*,** signifies a value of p<0.05 vs. sham-operated mice (SO) anduntreated infarcted mice (MI), respectively. Abbreviations are asfollows: MI-T, treated infarcted mice; LV, left ventricle and septum.FIG. 23A: To minimize the effects of cardiac hypertrophy in thesurviving myocardium and healing of the necrotic region with time oninfarct size, infarct dimension was measured by the loss of myocytes inthe left ventricle and septum. This measurement is independent fromreactive hypertrophy in the viable tissue and shrinkage of the necroticmyocardium with scar formation (87). FIG. 23B: Evaluation of LVhemodynamics is presented by data from LV end-diastolic pressure, LVdeveloped pressure, LV +dP/dt and LV −dP/dt. FIGS. 23C to 23H areconfocal micrographs which depict large infarcts of the left ventriclein an untreated mouse (23C and 23D) and in two treated mice (23E to23H). The area defined by a gate in FIGS. 23C, 23E and 23G (bars=1 mm)is illustrated at higher magnification in FIGS. 23D, 23F and 23H(bars=0.1 mm). In FIGS. 23C and 23D the lack of myocardial regenerationis illustrated by the accumulation of collagen type I and collagen typeIII (blue) in the infarcted region of the wall (arrows). Nuclei ofspared myocytes and inflammatory cells are apparent (green, PI). A smalllayer of viable myocytes is present in the subepicardium (red, cardiacmyosin). In FIGS. 23E to 23H, myocyte regeneration is illustrated by thered fluorescence of cardiac myosin antibody. Small foci of collagen typeI and type III (blue, arrowheads) are detected in the infarcted region.Nuclei are yellow-green (PI). Abbreviations are as follows: IS,interventricular septum; MI, myocardial infarct; RV, right ventricle.

FIG. 24 shows echocardiography results from a single mouse heart beforecoronary artery ligation and 15 days after ligation. Confocal microscopyshows a cross section of the same heart. FIG. 24A shows the baselineechocardiography results before coronary artery ligation. FIGS. 24B and24C show confocal microscopy at low (24B, bar=1 mm) and higher (24C,bar=0.1 mm) magnification of a cross section of the heart assessed inFIGS. 24A and 24D. Abbreviations used are as follows: RV, rightventricle; IS, interventricular septum; MI, myocardial infarct. FIG. 24Dshows the echocardiographic documentation of contractile function in thesame heart 15 days after infarction (arrowheads). FIG. 24E is a graphdepicting the ejection fraction with results reported as the mean±SD.*,** p<0.05 vs. sham-operated mice (SO) and untreated infarcted mice(MI), respectively. MI-T refers to treated infarcted mice.

FIGS. 25A-F shows confocal micrographs detailing properties ofregenerating myocytes. These properties are quantified in the graphs ofFIGS. 25G-J. FIGS. 25A and 25B depict enzymatically dissociated myocytesfrom the regenerating portion (25A) and surviving myocardium (25B) ofthe infarcted ventricle of a heart treated with growth factors. FIG. 25Ais stained to show small myocytes (red, cardiac myosin), bright nuclei(PI and BrdU), and blue nuclei (PI only). FIG. 25B shows large,hypertrophied myocytes (red, cardiac myosin), bright nuclei (PI andBrdU) and blue nuclei (PI only). In both FIGS. 25A and 25B, the barequals 50 μm. Mechanical properties of new (FIGS. 25C and 25D) andspared (FIGS. 25E and 25F) myocytes are shown after infarction in micetreated with growth factors. R refers to the relaxed state of theymyocytes, C is the contracted state. The effects of stimulation on cellshortening (G), velocity of shortening (H), time to peak shortening (I)and time to 50% re-lengthening (J) are depicted with results given for N(new small myocytes) and S (spared hypertrophied myocytes). Results arepresented as the mean±SD. * indicates a value of P<0.05 vs S.

FIG. 26 shows pairs of confocal micrographs showing various markers ofmaturing myocytes (FIGS. 26A to 26N, bar=10 μm). In FIGS. 26A to 26F,BrdU labeling of nuclei is shown in FIGS. 26A, 26C and 26E as greencoloration, and localization of nestin (FIG. 26B, red), desmin (FIG.26D, red), cardiac myosin (FIG. 26F, red) is shown in myocytes of tissuesections of regenerating myocardium. Nuclei are labeled by PI only inFIGS. 26B, 26D and 26F (blue), and by BrdU and PI together in FIGS. 26B,26D and 26F (bright). FIGS. 26G to 26N show the identification ofconnexin 43 (FIGS. 26G, 26H, 26K and 26L, yellow) and N-cadherin (FIGS.26I, 26J, 26M and 26N, yellow) in sections of developing myocardium(FIGS. 26G to 26J) and in isolated myocytes (FIGS. 26K to 26N). Myocytesare stained by cardiac myosin (FIGS. 26H, 26J, 26L and 26N, red) andnuclei by BrdU only (FIGS. 26G, 26I, 26K and 26M, green), PI only (FIGS.26H and 26J, blue) and by BrdU and PI together (FIGS. 26H, 26J, 26L and26N, bright).

FIG. 27 is a series of confocal micrographs showing newly formedcoronary vasculature. In 27A to 27D, arterioles are shown withTER-119-labeled erythrocyte membrane (green), PI staining of nuclei(blue), and α-smooth muscle actin staining of smooth muscle cell (red).In all micrographs, the bar equals 10 μm.

FIG. 28: Identification and growth of cardiac Lin⁻ c-kit^(POS) cellsobtained with immunomagnetic beads (a) and FACS (b). a,b, c-kit^(POS)cells in NSCM scored negative for cytoplasmic proteins of cardiac celllineages; nuclei are stained by PI (blue) and c-kit (green) by c-kitantibody. c-f, In DM at P1, cultured cells showed by purple fluorescencein their nuclei NRx2.5 (c), MEF2 (d), GATA-4 (e) and GATA-5 (f)labeling. g,h, Stem cells selected by NSCM and plated at low density (g)develop small individual colonies (h). Bar=10 μm.

FIG. 29: Self-renewal and multipotentiality of clonogenic cells. a,c-kit^(POS) cells in a clone: nuclei=blue, c-kit=green (arrowheads). b,Two of the 3 c-kit^(POS) cells (green, arrowheads) express Ki67 (purple,arrows) in nuclei (blue). c,d, Ki67 positive (c) metaphase chromosomes(red). d, metaphase chromosomes labeled by Ki67 and PI (purple) in ac-kit^(POS) cell (green). e-h, In the clone, the cytoplasm (red) of M(e), EC (f), SMC (g) and F (h) is stained by cardiac myosin, factorVIII, α-smooth muscle actin and vimentin, respectively. Nuclei=blue.Lin⁻ c-kit^(POS) cells (green, arrowheads) are present. Bar=10 μm.

FIG. 30: Clonogenic cells and spherical clones. a, Spherical clones(arrowheads) in suspension in NSCM. b, Cluster of c-kit^(POS) (green,arrowheads) and negative cells within the clone. Nuclei=blue. c,Spheroid with packed cell nuclei (blue) and large amount of nestin(red). d, Accumulation of non-degraded nestin (red) within the spheroid.Nuclei=blue. e, Spheroid plated in DM with cells migrating out of thesphere. f-h, M (f), SMC (g) and EC (h) migrating out of the spheroid anddifferentiating have the cytoplasm (red) stained respectively by cardiacmyosin, α-smooth muscle actin and factor VIII. Nuclei=blue. Bar=10 μm.

FIG. 31: Myocardial repair. a-c, Generating myocardium (a,b, arrowheads)in an infarcted treated rat (MI). New M=myosin (red);nuclei=yellow-green. Sites of injection (arrows). c, Myocardial scarring(blue) in an infarcted untreated rat. *Spared myocytes. d-n, Myocytes(h, myosin) and coronary vessels (k, EC=factor VIII; n, SMC=α-smoothmuscle actin) arising from the implanted cells are identified by BrdU(green) positive nuclei (g, j, m). Blue nuclei=PI (f, i, l). o-p,Myocytes at 20 days (p) are more differentiated than at 10 (o). Connexin43=green (arrowheads); Myosin=red; Nuclei=blue; BrdU=white. Bar=1 mm(a), 100 μM (b,c), 10 μM (d-p).

FIG. 32: Newly generated myocytes. a, enzymatically dissociated cellsfrom the repairing myocardial band. Cardiac myosin=red; Brdu=green;nuclei=blue. b-e, differentiation of new myocytes. Connexin 43=yellow(b,c); N-cadherin=yellow (d,e). Cardiac myosin=red; Brdu=green;nuclei=blue. Bar=10 μm.

FIG. 33: Mechanical properties of myocytes. a-d, new (N) and spared (S)myocytes obtained, respectively, from the regenerating and remainingmyocardium after infarction in treated rats; R=relaxed, C=contracted.e-h, effects of stimulation on cell shortening and velocity ofshortening of N (e,g) and S (f,h) myocytes.

FIG. 34: Primitive Cells in the Rat Heart. Section of left ventricularmyocardium from a Fischer rat at 22 months of age. A, Nuclei areillustrated by the blue fluorescence of propidium iodide (PI). B, Greenfluorescence documents c-kit positive cells. C, The combination of PIand c-kit is shown by green and blue fluorescence. The myocyte cytoplasmis recognized by the red fluorescence of α-sarcomeric actin antibodystaining. Confocal microscopy; bar=10 μm.

FIG. 35: FACS Analysis of c-kit^(POS) Cells. Bivariate distribution ofcardiac cells obtained from the left ventricle of a female Fischer 344rat showing the level of c-kit expression versus cellular DNA. The cellswere suspended at a concentration of 10⁶ cells/ml of PBS. Cellularfluorescence was measured with the ELITE ESP flow cytometer/cell sorter(Coulter Inc.) using an argon ion laser (emission at 488 nm) combinedwith a helium-cadmium laser, emitting UV light. Arrow indicates athreshold representing minimal c-kit level. For FACS analysis, cellswere incubated with r-phycoerythrin (R-PE)-conjugated rat monoclonalc-kit antibody (Pharmingen). R-PE isotype standard was used as anegative control.

FIG. 36: Scheme for Collection of Cardiac c-kit^(POS) Cells (A) andCulture of Cardiac c-kit^(POS) Cells in NSCM (B). A, Undifferentiatedcells expressing c-kit surface receptors are exposed to c-kit antibodyand subsequently to immunomagnetic beads coated by IgG antibody.c-kit^(POS) cells are collected with a magnet and cultured in NSCM. B,Immunomagnetic beads are attached on the surface of c-kit^(POS) cells(arrowheads). The absence of c-kit^(NEG) cells is apparent. Phasecontrast microscopy; bar=10 μm.

FIG. 37: c-kit Protein in Freshly Isolated Cells Collected withImmunomagnetic Beads. c-kit protein is shown by the green fluorescenceof c-kit antibody. Beads adherent to the cells are illustrated by redfluorescence. Blue fluorescence reflects PI labeling of nuclei. Thus,cells selected with beads were found to be c-kit^(POS). Confocalmicroscopy; bar=10 μm.

FIG. 38: Transcription Factors of Cardiomyocyte Differentiation. Afterremoval of the beads, or immediately after FACS separation, smears weremade and cells were stained for the detection of NRx2.5, MEF2 andGATA-4. Blue fluorescence in panels A-C corresponds to PI labeling ofnuclei. Purple fluorescence in nuclei reflects the expression of NRx2.5(A), MEF2 (B) and GATA-4 (C). Confocal microscopy; bar=10 μm.

FIG. 39: c-kit^(POS) Cells and Transcription Factors of Skeletal MuscleDifferentiation. Panels A-C shows c-kit^(POS) cells (green fluorescence,c-kit antibody; blue fluorescence, PI labeling). Panels D-F illustratepositive controls (C2C12 myoblast cell line) for MyoD (D), myogenin (E),and Myf5 (F) by green fluorescence within nuclei (red fluorescence, PIlabeling). c-kit^(POS) cells were negative for these skeletal muscletranscription factors. Confocal microscopy; bar=10 μm.

FIG. 40: Growth of c-kit^(POS) Cells in Differentiating Medium (DM).Monolayer of confluent cells obtained from plating c-kit positive cells.Immunomagnetic beads were removed by gentle digestion of the cells withDNase I. This procedure degraded the short DNA linker between the beadand the anti-IgG antibody. Phase contrast microscopy; bar=20 μm.

FIG. 41: Cycling Cell Nuclei in DM. Ki67 (purple fluorescence) isexpressed in the majority of nuclei contained in the field. Bluefluorescence reflects PI labeling of nuclei. Confocal microscopy;bars=10 μm.

FIG. 42: Growth Rate of c-kit^(POS)-Derived Cells. Exponential growthcurves of cells at P2 and P4; t_(D), time required by the cells todouble in number. Each point corresponds to 5 or 6 independentdeterminations. Vertical bars, SD.

FIG. 43: Identification and Growth of Cardiac Lin⁻ c-kit^(POS) Cells. InDM at P3, the cytoplasm (green) of M (A), EC (B), SMC (C) and F (D) isstained by cardiac myosin, factor VIII, α-smooth muscle actin andvimentin (factor VIII negative), respectively. Nuclei=red. Confocalmicroscopy; bars=10 μm.

FIG. 44: Cytoplasmic Markers of Neural Cells. Panels A-C shows cells inDM at P1 (red fluorescence, α-sarcomeric actin; blue fluorescence, PIlabeling). Panels D-F illustrate positive controls for MAP1b (D,neuron2A cell line), nerofilament 200 (E, neuron2A cell line), and GFAP(F, astrocyte type III, clone C8-D30) by green fluorescence in thecytoplasm (blue fluorescence, PI labeling). c-kit^(POS)-derived cellswere negative for these neural proteins. Confocal microscopy; bar=10 μm.

FIG. 45: Cytoplasmic Markers of Fibroblasts. Panels A-C shows smallcolonies of undifferentiated cells in NSCM (green fluorescence, c-kit;blue fluorescence, PI labeling). Panels D-F illustrate positive controls(rat heart fibroblasts) for fibronectin (D), procollagen type I (E), andvimentin (F) by red fluorescence in the cytoplasm (blue fluorescence, PIlabeling). c-kit^(POS)-derived cells were negative for these fibroblastproteins. Confocal microscopy; bar-10 μm.

FIG. 46: FACS-Isolated c-kit^(POS) Cells: Multipotentiality ofClonogenic Cells. In a clone, the cytoplasm (red) of M (A), EC (B), SMC(C) and F (D) is stained by cardiac myosin, factor VIII, α-smooth muscleactin and vimentin, respectively. Blue fluorescence, PI labeling ofnuclei. Lin⁻ c-kit^(POS) cells (green fluorescence, arrowheads) arepresent. Confocal microscopy; bar=10 μm.

FIG. 47: Cardiac Cell Lineages in Early Differentiation. A,B, Expressionof nestin alone (green fluorescence) in the cytoplasm of cells in earlydifferentiation. C,D, Expression of nestin (green, C) and cardiac myosin(red, D) in developing myocytes (arrowheads). E,F, Expression of nestin(green, E) and factor VIII (red, F) in developing endothelial cells(arrowheads). G,H, Expression of nestin (green, G) and α-smooth muscleactin (red, H) in developing smooth muscle cells (arrowheads). Confocalmicroscopy; bars=10 μm.

FIG. 48: Infarct Size and Myocardial Repair. A, At 10 days, coronaryartery occlusion resulted in the loss of 49% and 53% of the number ofmyocytes in the left ventricle of untreated (MI) and treated (MI-T)rats, respectively. At 20 days, coronary artery occlusion resulted inthe loss of 55% and 70% of the number of myocytes in the left ventricleof untreated (MI) and treated (MI-T) rats, respectively. SO,sham-operated animals. *P<0.05 vs SO. ^(†)P<0.05 vs MI. B, Percentage ofnewly formed myocardium within the infarcted region of the wall at 10and 20 days (d) after coronary artery occlusion in animals treated withcell implantation (MI-T). *P<0.05 vs 10 d. C,D, The amount of newmyocardium formed (F) at 10 and 20 days by cell implantation wasmeasured morphometrically (solid bar). The remaining (R) and lost (L)myocardium after infarction is depicted by hatched bar and crosshatchedbar, respectively. The generated tissue (F) increased the remainingmyocardium (R+F) and decreased the lost myocardium (L-F) by the sameamount. As a consequence, cardiac repair reduced infarct size in bothgroups of rats treated with cell implantation. Results are mean±SD.*P<0.05 vs MI. ^(†)P<0.05 vs Lo and Fo in MI-T.

FIG. 49: Myocardial Repair. A,B, Bands of regenerating myocardium in twoinfarcted treated hearts. Red fluorescence corresponds to cardiac myosinantibody staining of newly formed myocytes. Yellow-green fluorescencereflects PI labeling of nuclei. Blue fluorescence (arrowheads)illustrates small foci of collagen accumulation within the infarctedregion of the wall. Confocal microscopy; bar=100 μm.

FIG. 50: Neoformation of Capillaries. The differentiation of implantedcells in capillary profiles was identified by BrdU labeling ofendothelial cells. A, PI labeling of nuclei (blue); B, BrdU labeling ofnuclei (green); C, Capillary endothelium (red) and endothelial cellnuclei labeled by BrdU (blue and green). Confocal microscopy; bar=10 μm.

FIG. 51: Volume Composition of Regenerating Myocardium. During theinterval from 10 to 20 days, the volume fraction of myocytes (M),capillaries (Cap) and arterioles (Art) increased 25%, 62% and 140%,respectively. Conversely, the volume percent of collagen type I (C-I)and collagen type III (C-III) decreased 73% and 71%, respectively.Results are mean±SD. *P<0.05 vs 10 days.

FIG. 52: Cell Proliferation in the Regenerating Myocardium. During theinterval from 10 to 20 days, the fraction of myocytes (M), endothelialcells (EC) and smooth muscle cells (SMC) labeled by Ki67 decreased 64%,63% and 59% respectively. Results are mean±SD. *P<0.05 vs 10 days.

FIG. 53: Identification of Regenerating Myocytes by BrdU Labeling. A,D,Nuclei are illustrated by the blue fluorescence of PI. B,E, Greenfluorescence documents BrdU labeling of nuclei. C,F, Myocyte cytoplasmis recognized by the red fluorescence of α-cardiac actinin (C) orα-sarcomeric actin (F). In new myocytes, dark and light bluefluorescence reflects the combination of PI and BrdU labeling of myocytenuclei (C,F). Confocal microscopy; bar=10 μm.

FIG. 54: Effects of Time on Number and Volume of Newly Formed Myocytes.During the interval from 10 to 20 days, developing myocytes increasedsignificantly in size. However, cell number remained essentiallyconstant. The size distribution was wider at 20 than at 10 days.

FIG. 55: Effects of Time on the Development of Newly Formed CoronaryVasculature. The numerical density of newly formed arterioles (Art) andcapillaries (Cap) increased significantly during the interval from 10 to20 days. Results are mean±SD. *P<0.05 vs 10 days.

FIG. 56: Spared Myocytes in the Infarcted Ventricle. A,B, Large,hypertrophied myocytes isolated from the remaining viable tissue of theleft ventricle and interventricular septum. Red fluorescence correspondsto cardiac myosin antibody staining and blue fluorescence to PIlabeling. Yellow fluorescence at the edges of the cells reflectsconnexin 43 (A) and N-cadherin (B). Confocal microscopy; bar=10 μm.

FIG. 57: Cell Implantation and Echocardiography. Myocardial regenerationattenuated ventricular dilation (A), had no effect on the thickness ofthe surviving portion of the wall (B), increased the thickness of theinfarcted region of the ventricle (C) and improved ejection fraction(D). SO=sham-operated; MI=untreated infarcts; MI-T=treated infarcts.Results are mean±SD. *P<0.05 vs SO; **P<0.05 vs MI.

FIG. 58: Echocardiographic Tracing. Two-dimensional images and M-modetracings of an untreated infarcted rat (A,B,C) and a treated infarctedrat (D,E,F). Panels A and D correspond to baseline conditions beforecoronary artery occlusion. The reappearance of contraction is evident inpanel F (arrowheads).

FIG. 59. Ventricular Function and Wall Stress. Cell implantationimproved ventricular function (A-D) and attenuated the increase indiastolic wall stress (E) after infarction. SO=sham-operated;MI=untreated infarcts; MI-T=treated infarcts; LVEDP=left ventricularend-diastolic pressure; LVDP=left ventricular developed pressure;+dP/dt=rate of pressure rise; −dP/dt=rate of pressure decay. Results aremean±SD. *P<0.05 vs SO; **P<0.05 vs MI.

FIG. 60: Cell Implantation in Normal Myocardium. BrdU labeled cellsobtained at P2 were injected in sham-operated rats. Twenty days later,only a few undifferentiated cells were identified. A,C, Greenfluorescence documents BrdU labeling of nuclei. B,D, Myocyte cytoplasmis recognized by the red fluorescence of α-sarcomeric actin. Nuclei areillustrated by the blue fluorescence of PI. In injected cells(arrowheads), bright blue fluorescence reflects the combination of PIand BrdU labeling (B,D). Confocal microscopy; bar=10 μm.

FIGS. 61 and 62. Migration and invasion assays. Results in are reportedas the mean±SD. * indicates a statistical significant difference, i.e.P<0.05, from cells not exposed to the growth factor.

FIG. 63. Matrix metalloproteinase activity assay. Digital photograph ofthe resulting gel from gelatin zymography,

FIG. 64. Graphs of primitive cells expressing growth factor receptors.The distribution of c-met and IGF-IR on c-kit^(POS) and MDR1^(POS) cellsin the various regions of the heart in sham-operated (SO),infarcted-treated (Treated) and infarcted-untreated (Untreated) micesacrificed 7-8 hours after surgery and 2-3 hours after theadministration of growth factors (Treated) or saline (SO; Untreated) isshown. These measurements include all c-kit^(POS) and MDR1^(POS) cells,independently of ongoing apoptosis. Abbreviations used are as follows:A, atria; LV, left ventricle; R, viable myocardium remote from theinfarct; B, viable myocardium bordering the infarct; I, non-viableinfarcted myocardium. All results are reported as the mean±SD.

FIG. 65. Graphs showing the location of cycling primitive cells. Thepercentage of viable Ki67 labeled c-kit^(POS) and MDR1^(POS) cells inthe various regions of the heart in sham-operated (SO),infarcted-treated (Treated) and infarcted-untreated (Untreated) micesacrificed 7-8 hours after surgery and 2-3 hours after theadministration of growth factors (Treated) or saline (SO; Untreated) ispresented. Abbreviations used are as follows: A, atria; LV, leftventricle; R, viable myocardium remote from the infarct; B, viablemyocardium bordering the infarct; I, non-viable infarcted myocardium.Results presented are means±SD.

FIG. 66. Graphs showing the frequency distribution of DNA content innon-cycling (solid line) and cycling (broken line; Ki67 positive nuclei)myocytes. Both new and old myocytes showed an amount of chromatincorresponding to 2n chromosomes. A DNA content greater than 2n wasrestricted to cycling nuclei. The measured non-cycling nuclei displayeda fluorescence intensity comparable to that of diploid lymphocytes.Sampling included 600 new myocytes, 1,000 old myocytes and 1,000lymphocytes.

FIG. 67. Graphs showing the effects of myocardial infarction on theanatomy of the heart and diastolic load. Results are presented as themean±SD. *,** indicate a value of p<0.05 vs. sham-operated mice (SO) anduntreated infarcted mice (MI). MI-T refers to treated infarcted mice.

FIG. 68. Graph showing the frequency distribution of myocyte sizes. Thevolume of newly generated myocytes was measured in sections stained withdesmin and laminin antibodies and PI. Only longitudinally oriented cellswith centrally located nuclei were included. The length and diameteracross the nucleus were collected in each myocyte to compute cellvolume, assuming a cylindrical shape. Four hundred cells were measuredin each heart.

FIG. 69: Graph showing cardiac repair. On the basis of the volume of LVin sham-operated (SO) mice and infarct size, 42% in untreated mice (MI)and 67%. in treated mice (MI-T), the volume of myocardium destined toremain (R) and destined to be lost (L) was computed in the two groups ofinfarcted mice (FIG. 9). The volume of newly formed myocardium (F) wasmeasured quantitatively in treated mice. Myocardial regenerationincreased the volume of remaining myocardium (R+F) and decreased thevolume of lost myocardium (L-F) by the same amount. Therefore, infarctsize in treated mice was reduced by 15%.

FIG. 70A-J. Photomicrographs of the isolation and culture of humancardiac progenitor cells. Seeding of human myocardial samples (MS) forthe outgrowth of cardiac cells (A and B); at ˜2 weeks, clusters of cells(C; vimentin, green) surround the centrally located explant. Cellspositive for c-kit (D; green, arrows), MDR1 (E; magenta, arrows) andSca-1-like-protein (F; yellow, arrows) are present. Some nuclei expressGATA4 (E; white) and MEF2C (F; magenta). Myocytes (G; α-sarcomericactin, red), SMCs (H; α-SM actin, magenta), ECs (I; von Willebrandfactor, yellow) and a cell positive for neurofilament 200 (J; white)were detected in the outgrowing cells together with smallc-kit^(POS)-cells (green, arrows).

FIG. 71. Growth Properties of Human Cardiac Progenitor Cells. Sortedc-kit^(POS)-cells (green) are lineage negative (arrowheads) or expressGATA4 in their nuclei (A; white). Several c-kit^(POS)-cells are labeledby Ki67 (B; red, arrowheads) and one expresses p16^(INK4a) (C; yellow,arrowhead). D: By FACS analysis, cardiac progenitor cells (P1-P3) werenegative for CD34, CD45, and CD133, and 52 percent were positive forCD71. Isotype, black; antigen, red. As shown by immunocytochemistry,c-kit^(POS)-cells (E; green) express nestin in their cytoplasm (F; red).The colocalization of c-kit and nestin is shown in panel G (yellow). H:Individual c-kit^(POS)-cells (green) plated in single wells; theformation of clones from single founder c-kit^(POS)-cells is shown inpanel I. J: C-kit^(POS)-cells in differentiating medium form myocytesα-sarcomeric actin, red), SMCs (α-SM actin, magenta) and ECs (vonWillebrand factor, yellow).

FIG. 72. Human C-kit^(POS)-Cells Regenerate the Infarcted Myocardium.FIGS. 72A-C are photomicrographs. FIG. 72A depicts an infarcted heart inan immunodeficient mouse injected with human c-kit^(POS)-cells andsacrificed 21 days later. The large transverse section shows a band ofregenerated myocardium (arrowheads) within the infarct (MI). BZ, borderzone. The two areas included in the rectangles are illustrated at highermagnification in the panels below. The localization of the Alu probe inthe newly formed myocytes is shown by green fluorescence dots in nuclei.Newly formed myocytes are identified by α-sarcomeric actin (red;arrowheads). Myocyte nuclei are labeled by propidium-iodide (blue).Asterisks indicate spared myocytes. FIGS. 72B and C: Examples ofregenerated myocardium (arrowheads), 21 and 14 days after infarction andthe injection of human cells, in an immunodeficient mouse (B) and in animmunosuppressed rat (C). Newly formed myocytes are identified byα-sarcomeric actin (red). Myocyte nuclei are labeled by Alu (green) andBrdU (white). Asterisks indicate spared mouse and rat myocytes. FIG. 72Dincludes a hematoxylin and eosin (H&E) stained section shows samplingprotocol: Also provided are photographs of gels used in detection ofhuman DNA in the regenerated infarcted myocardium; human blood (Human)and intact rat myocardium (Rat) were used as positive and negativecontrols. The signal for rat MLC2v DNA in the infarcted myocardiumreflects the presence of spared myocytes in the subendocardial and/orsubepicardial region of the wall (see FIG. 72A-C). FIGS. 72E and F, andG and H are photomicrographs that illustrate the same fields. Newlyformed myocytes (E-H; troponin I, qdot 655, red) express GATA4 (E; qdot605, white) and MEF2C (G: qdot 605, yellow). Laminin: qdot 525, white.Myocyte nuclei are labeled by Alu (F and H; green). Connexin 43 (I; qdot605, yellow, arrowheads) and N-cadherin (J; qdot 605, yellow,arrowheads) are detected between developing myocytes by cardiac myosinheavy chain (MHC; qdot 655, red). These structures are positive for Alu.Sarcomere striation is apparent in some of the newly formed myocytes(E-J). FIGS. 72A, B, E, F, I, J: infarcted immunodeficient mice, 21 daysafter cell implantation. FIGS. 72C, D, G, H: infarcted immunosuppressedrats, 14 days after cell implantation.

FIG. 73. FIGS. 73A-F and H are photomicrographs depicting coronarymicrovasculature and cell fusion. Human coronary arterioles with layersof SMCs (A-C; α-SM actin, qdot 655, red). The endothelial lining of thearteriole in C is shown in D by von Willebrand factor (qdot 605,yellow). FIGS. 73E and F: Human capillaries (von Willebrand factor, qdot605, yellow). Nuclei are labeled by Alu (A-F; green). FIG. 73G depictsgraphs showing the extent of vasculogenesis in the human myocardium;results are mean±SD. FIG. 73H: Human X-chromosomes (white dots;arrowheads) in regenerated myocytes and vessels in the mid-region of theinfarct. Mouse X-chromosomes (magenta dots; arrows) are present inmyocytes located at the border zone in proximity of regenerated humanmyocytes. Nuclei exhibit no more than two human X-chromosomes excludingcell fusion.

FIG. 74. Myocardial Regeneration and Cardiac Function. FIGS. 74A-C arephotomicrographs showing a transmural infarct. The transmural infract ina non-treated rat is shown in FIG. 74A (arrowheads); areas in therectangles are shown at higher magnification in the lower panels. Deadmyocytes without nuclei (dead, α-sarcomeric actin, red). Connectivetissue cell nuclei (blue). The echocardiogram shows the lack ofcontraction in the infarcted region of the wall (arrows). FIG. 74B:Transmural infarct (arrowheads) in a treated rat; the area included inthe rectangle is shown at higher magnification in the lower panels.Human myocytes (α-sarcomeric actin, red) are labeled by Alu (green). Theechocardiogram shows the presence of contraction in the infarcted regionof the wall (arrowheads). FIG. 74C shows at higher magnification anothertransmural infarct (arrowheads) in a treated rat in which regeneratedhuman myocytes α-sarcomeric actin, red) in the mid-region of the infarctare labeled by Alu (green). The echocardiogram shows the presence ofcontraction in the infarcted region of the wall (arrowheads). FIG. 74Dis a graph illustrating that myocardial regeneration in treated rathearts increased ejection fraction. Echocardiography in mice was usedonly for detection of contraction in treated mice as previouslyindicated. ¹³ FIGS. 74E and F are graphs showing the effects ofmyocardial regeneration on the anatomy and function of the infarctedheart. Data are mean±SD. *,^(†) indicate a difference, P<0.05, versus SOand MI, respectively.

FIG. 75. A graph showing the multipotentiality of C-kit^(POS)-Cells.C-kit^(POS)-cells at various passages (P) are capable of acquiring thecardiac commitment (GATA4-positive) and generating myocytes(α-sarcomeric actin-positive), SMCs (α-SM actin-positive) and ECs (vonWillebrand factor-positive). Results are mean±SD.

FIG. 76. Photomicrographs showing characteristics of the regeneratedhuman myocardium. Regenerated myocytes and coronary arterioles afterinfarction and implantation of human cells; newly formed myocytes (A;cardiac myosin heavy chain, red), SMCs (B, D, E; α-SM actin, magenta)and ECs (C, F, G; von Willebrand factor, yellow) are present. Thedistribution of laminin between myocytes is shown by white fluorescence(A). Panels D and E, and panels F and G illustrate the same fields.Dispersed SMCs (D and E; arrows) and ECs (F and G; arrows) are present.GATA6 (D; red dots in nuclei) and Ets1 (F; magenta dots in nuclei) aredetected in SMCs and ECs, respectively. These structures are positivefor the Alu probe (green). A-G: infarcted immunodeficient mice, 21 daysafter cell implantation.

FIG. 77. Graphs showing the volume of human myocytes. Size distributionof newly formed human myocytes in infarcted mice and rats.

FIG. 78. Photomicrographs showing functionally competent humanmyocardium. Transmural infarct (arrowheads) in a treated mouse in whichregenerated human myocytes in the mid-region of the infarct are positivefor α-sarcomeric actin (red). Human nuclei are labeled by the Alu probe(green). The echocardiogram shows the presence of contraction in theinfarcted region of the wall (arrowheads).

FIG. 79. Homing and engraftment of activated CSCs. FIG. 79A, Site ofinjection of GF-activated clonogenic CSCs expressing EGFP (green) at 24hours after infarction. FIGS. 79B-D, Some activated-CSCs are TdT labeledat 12 hours (B; magenta, arrowheads) and several are positive for thecell cycle protein Ki67 at 24 hours (C; white, arrows). D, Rates ofapoptosis and proliferation in activated-CSCs at 12, 24 and 48 hoursafter injection. Values are mean±s.d. *P<0.05 versus 12 hours; **P<0.05versus 24 hours. FIGS.79E-G, Connexin 43, N-cadherin, E-cadherin andL-selectin (white) are expressed between EGFP-positive cardiacprogenitor cells (arrowheads), and between EGFP-positive cardiacprogenitor cells and EGFP-negative recipient cells (arrows) at 48 hoursafter infarction and cell injection; myocytes are stained byα-sarcomeric actin (red) and fibroblasts by procollagen (yellow). FIG.79H, Apoptotic EGFP-positive cells (TdT, magenta, arrows) do not expressL-selectin (white). FIG. 79I, Site of injection of GF-activatedclonogenic CSCs, EGFP positive (green), at one month after implantationin an intact non-infarcted heart. Nuclei, PI (blue).

FIG. 80. Vessel regeneration. a, The epimyocardium of aninfarcted-treated heart at 2 weeks shows 3 newly formed coronaryarteries (upper panel, EGFP, green) located within the spared myocardium(arrow) and at the border zone (BZ, arrowheads). A branching vessel isalso visible (open arrow). The colocalization of EGFP and α-smoothmuscle actin (α-SMA) is shown in the lower panel (orange). The minordiameter of the vessels is indicated. The vessel with a diameter of 180μm has an internal elastic lamina (inset; IEL, magenta). Preexistingcoronary branches are EGFP-negative (upper panel) and α-SMA-positive(lower panel, red, asterisks). A cluster of EGFP-positive cells ispresent at the site of injection (SI). Myocytes are labeled byα-sarcomeric actin (α-SA). b, The spared myocardium of the epicardiallayer at 2 weeks contains several large regenerated coronary arteries(upper panels, EGFP, green), which express EGFP and α-SMA (lower panels,EGFP-α-SMA, orange). c, The infarcted myocardium in the mid-region ofthe wall shows regenerated intermediate and small-sized coronaryarteries (upper panels, EGFP, green), which express EGFP and α-SMA(lower panels, EGFP-α-SMA, orange). d, Magnitude of vessel formation inthe heart. Values are mean±s.d. e, SMCs and ECs in regenerated coronaryarterioles exhibit at most two X-chromosomes. EGFP-α-SMA, orange.X-chromosomes, white dots.

FIG. 81. The newly formed coronary vessels are functionally competent.FIGS. 81A-F, Large coronary arteries and branches located in the viable(A,F) and infarcted (B-E) myocardium of the epicardial layer of treatedrats at 2 weeks (A-C) and one month (D-F) containrhodamine-labeled-dextran (red), and possess EGFP-positive wall (green).Collagen (blue) is abundant in the infarct (B-E) and mostlyperi-vascular in the surviving myocardium (A,F). The vessel diameter isindicated. The vessel and its branches in FIG. 81E are surrounded byEGFP-positive cells, which are located within the infarcted myocardium.FIG. 81F documents the functional integration of newly formed vessels(EGFP-positive wall, green) with resident vessels (EGFP-negative wall).The white circles delimit the sites of anastomosis. FIG. 81G,Ventricular anatomy and infarct size. FIG. 81H, Ventricular function.Left ventricular end-diastolic pressure, LVEDP; LV developed pressure,LVDP. Values are mean±s.d. Untreated myocardial infarcts, MI. Treatedmyocardial infarcts, MI-T.

FIG. 82. Experimental protocol. Permanent coronary occlusion was inducedby ligation of the left anterior descending coronary artery (LAD). Twoforms of treatment were employed: 1. Injection of a total number of80,000-100,000 clonogenic EGFP^(POS)-c-kit^(POS)-CSCs(non-activated-CSCs); 2. Injection of EGFP^(POS)-c-kit^(POS)-CSCspretreated in vitro with GFs per 2 hours (activated-CSCs) prior to theirimplantation in vivo. Injections were performed at multiple sites (blackdots) above, laterally and below the ligature, distant from the borderzone (BZ). MI, myocardial infarction.

FIG. 83. Cardiac stem cell death. Rates of apoptosis and proliferationin CSCs at 12, 24 and 48 hours after injection. Values are mean±s.d.

FIG. 84. Vessel regeneration. a, The spared myocardium of the epicardiallayer at one month contains several large regenerated coronary arteries(upper panels, EGFP, green), which express EGFP and α-SMA (lower panels,EGFP-α-SMA, orange). b, The infarcted myocardium in the mid-region ofthe wall shows regenerated large, intermediate and small-sized coronaryarteries (upper panels, EGFP, green), which express EGFP and α-SMA(lower panels, EGFP-α-SMA, orange). c, Regenerated capillaries in theinfarcted myocardium express EGFP (green) and von are labeled byEC-specific lectin (white).

DETAILED DESCRIPTION

The present invention provides methods and/or pharmaceutical compositioncomprising a therapeutically effective amount of somatic stem cellsalone or in combination with a cytokine selected from the groupconsisting of stem cell factor (SCF), granulocyte-colony stimulatingfactor (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), stromal cell-derived factor-1, steel factor, vascularendothelial growth factor, macrophage colony stimulating factor,granulocyte-macrophage stimulating factor, hepatocyte growth factor(HGF), insulin-like growth factor (IGF-1) or Interleukin-3 or anycytokine capable of the stimulating and/or mobilizing stem cells.Cytokines may be administered alone or in combination or with any othercytokine or pharmaceutical agent capable of: the stimulation and/ormobilization of stem cells; the maintenance of early and latehematopoiesis (see below); the activation of monocytes (see below),macrophage/monocyte proliferation; differentiation, motility andsurvival (see below); treatment of cardiac or vascular conditions; and apharmaceutically acceptable carrier, diluent or excipient (includingcombinations thereof).

The invention also provides methods and/or pharmaceutical compositionscomprising a therapeutically effective amount of one or more cytokinesfor causing the migration and/or proliferation of cardiac stem cells orcardiac primitive cells into circulatory tissue or muscle tissue orcirculatory muscle tissue, e.g., cardiac tissue, such as the heart orblood vessels—e.g., veins, arteries, that go to or come from the heartsuch as veins and arteries directly connected or attached or flowinginto the heart, for instance the aorta.

In a preferred aspect, the methods and/or compositions, includingpharmaceutical compositions, comprise effective amounts of two or morecytokines. More specifically, the methods and/or compositions preferablycomprise effective amounts of hepatocyte growth factor and insulin-likegrowth factor-1.

The cytokines in the pharmaceutical composition of the present inventionmay also include mediators known to be involved in the maintenance ofearly and late hematopoiesis such as IL-1 alpha and IL-1 beta, IL-6,IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin,erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growthfactor, tumor necrosis factor alpha, leukemia inhibitory factor,transforming growth factors beta 1 and beta 3; and macrophageinflammatory protein 1 alpha), angiogenic factors (fibroblast growthfactors 1 and 2, vascular endothelial growth factor) and mediators whoseusual target (and source) is the connective tissue-forming cells(platelet-derived growth factor A, epidermal growth factor, transforminggrowth factors alpha and beta 2, oncostatin M and insulin-like growthfactor-1), or neuronal cells (nerve growth factor) (Sensebe, L., et al.,Stem Cells 1997; 15:133-43), VEGF polypeptides that are present inplatelets and megacaryocytes (Wartiovaara, U., et al., Thromb Haemost1998; 80:171-5; Mohle, R., Proc Natl Acad Sci USA 1997; 94:663-8) HIF-1,a potent transcription factor that binds to and stimulates the promoterof several genes involved in responses to hypoxia, endothelial PASdomain protein 1 (EPAS 1), monocyte-derived cytokines for enhancingcollateral function such as monocyte chemotactic protein-1 (MCP-1).

In an additionally preferred aspect, the methods and/or compositions,including pharmaceutical compositions, comprise effective amounts of twoor more cytokines in combination with an appropriate pharmaceuticalagent useful in treating cardiac and/or vascular conditions.

In a preferred aspect, the pharmaceutical composition of the presentinvention is delivered via injection. These routes for administration(delivery) include, but are not limited to subcutaneous or parenteralincluding intravenous, intraarterial, intramuscular, intraperitoneal,intramyocardial, transendocardial, trans-epicardial, intranasaladministration as well as intrathecal, and infusion techniques. Hence,preferably the pharmaceutical composition is in a form that is suitablefor injection.

When administering a therapeutic of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired.

The pharmaceutical composition of the present invention, e.g.,comprising a therapeutic compound, can be administered to the patient inan injectable formulation containing any compatible carrier, such asvarious vehicles, adjuvants, additives, and diluents; or the compoundsutilized in the present invention can be administered parenterally tothe patient in the form of slow-release subcutaneous implants ortargeted delivery systems such as monoclonal antibodies, iontophoretic,polymer matrices, liposomes, and microspheres.

The pharmaceutical composition utilized in the present invention can beadministered orally to the patient. Conventional methods such asadministering the compounds in tablets, suspensions, solutions,emulsions, capsules, powders, syrups and the like are usable. Knowntechniques which deliver the compound orally or intravenously and retainthe biological activity are preferred.

In one embodiment, a composition of the present invention can beadministered initially, and thereafter maintained by furtheradministration. For instance, a composition of the invention can beadministered in one type of composition and thereafter furtheradministered in a different or the same type of composition. Forexample, a composition of the invention can be administered byintravenous injection to bring blood levels to a suitable level. Thepatient's levels are then maintained by an oral dosage form, althoughother forms of administration, dependent upon the patient's condition,can be used.

It is noted that humans are treated generally longer than the mice orother experimental animals which treatment has a length proportional tothe length of the disease process and drug effectiveness. The doses maybe single doses or multiple doses over a period of several days, butsingle doses are preferred. Thus, one can scale up from animalexperiments, e.g., rats, mice, and the like, to humans, by techniquesfrom this disclosure and documents cited herein and the knowledge in theart, without undue experimentation.

The treatment generally has a length proportional to the length of thedisease process and drug effectiveness and the patient being treated.

The quantity of the pharmaceutical composition to be administered willvary for the patient being treated. In a preferred embodiment,2×10⁴-1×10⁵ stem cells and 50-500 μg/kg per day of a cytokine wereadministered to the patient. While there would be an obvious sizedifference between the hearts of a mouse and a human, it is possiblethat 2×10⁴-1×10⁵ stem cells would be sufficient in a human as well.However, the precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, size of the infarct, and amount of time sincedamage. Therefore, dosages can be readily ascertained by those skilledin the art from this disclosure and the knowledge in the art. Thus, theskilled artisan can readily determine the amount of compound andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or cytokine(s)) are present inan amount of 0.001 to 50 wt % solution in phosphate buffered saline, andthe active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt %or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %,and most preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD₅₀ in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Additionally, one of skill in the art would be able to ascertain withoutundue experimentation the appropriate pharmaceutical agent to be used incombination with one or more cytokines; and, one of skill in the artwould be able to make the precise determination of what would beconsidered an effective dose based on factors individual to eachpatient, including their size, age, size of the infarct, and amount oftime since damage. Therefore, dosages can be readily ascertained bythose skilled in the art from this disclosure and the knowledge in theart.

Examples of compositions comprising a therapeutic of the inventioninclude liquid preparations for orifice, e.g., oral, nasal, anal,vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar,gingival, olfactory or respiratory mucosa) etc., administration such assuspensions, syrups or elixirs; and, preparations for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as sterile suspensions oremulsions. Such compositions may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose or the like. The compositions can also be lyophilized.The compositions can contain auxiliary substances such as wetting oremulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Standard texts, such as “REMINGTON′S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or,a dose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like (e.g., fortransdermal administration) and will typically contain a sufficientamount of a thickening agent so that the viscosity is from about 2500 to6500 cps, although more viscous compositions, even up to 10,000 cps maybe employed. Viscous compositions have a viscosity preferably of 2500 to5000 cps, since above that range they become more difficult toadminister. However, above that range, the compositions can approachsolid or gelatin forms which are then easily administered as a swallowedpill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with mucosa, such as the lining of the stomach or nasalmucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels normally contain a major amount of water(preferably purified water) in addition to the active compound. Minoramounts of other ingredients such as pH adjusters (e.g., a base such asNaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, (e.g., methylcellulose),colors and/or flavors may also be present. The compositions can beisotonic, i.e., they can have the same osmotic pressure as blood andlacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert with respect tothe active compound. This will present no problem to those skilled inchemical and pharmaceutical principles, or problems can be readilyavoided by reference to standard texts or by simple experiments (notinvolving undue experimentation), from this disclosure and the documentscited herein.

The inventive compositions of this invention are prepared by mixing theingredients following generally accepted procedures. For example theselected components may be simply mixed in a blender, or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity. Generally the pH may be from about 3 to 7.5.Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the composition form used for administration(e.g., solid vs. liquid). Dosages for humans or other mammals can bedetermined without undue experimentation by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

Suitable regimes for initial administration and further doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the knowledge in the art.

The pharmaceutical compositions of the present invention are used totreat cardiovascular diseases, including, but not limited to,atherosclerosis, ischemia, hypertension, restenosis, angina pectoris,rheumatic heart disease, congenital cardiovascular defects and arterialinflammation and other diseases of the arteries, arterioles andcapillaries or related complaint. Accordingly, the invention involvesthe administration of stem cells as herein discussed, alone or incombination with one or more cytokine, as herein discussed, for thetreatment or prevention of any one or more of these conditions or otherconditions involving weakness in the heart, as well as compositions forsuch treatment or prevention, use of stem cells as herein discussed,alone or in combination with one or more cytokine, as herein discussed,for formulating such compositions, and kits involving stem cells asherein discussed, alone or in combination with one or more cytokine, asherein discussed, for preparing such compositions and/or for suchtreatment, or prevention. And, advantageous routes of administrationinvolves those best suited for treating these conditions, such as viainjection, including, but are not limited to subcutaneous or parenteralincluding intravenous, intraarterial, intramuscular, intraperitoneal,intramyocardial, transendocardial, trans-epicardial, intranasaladministration as well as intrathecal, and infusion techniques.

In one embodiment of the invention, there are provided methods andcompositions for the treatment of vasculature disorders or disease,including the occlusion or blockage of a coronary artery or vessel. Thepresent invention provides methods and compositions that can be used forsuch therapeutic treatment as an alternative to, or in combination with,cardiac bypass surgery. The present invention provides for theisolation, expansion, activation, and implantation or delivery ofactivated stem cells, preferably activated cardiac stem cells, to anarea of the vasculature in need thereof. Such delivery or implantationcan be accomplished by any of the methods described herein or which areknown to those of skill in the art, including, but not limited to, theuse of a NOGA catheter system such that visualization of the area to betreated is possible, and the therapeutic is delivered via a retractableneedle associated with such catheter system. One of skill in the artwill recognize other useful methods of delivery or implantation whichcan be utilized with the present invention, including those described inDawn, 2005, the contents of which are incorporated herein in theirentirety.

In a further embodiment, cardiac tissue is harvested from a patient inneed of therapeutic treatment for one of the cardiac or vasculatureconditions described herein. The present invention provides for theisolation of stem cells, preferably cardiac stem cells, more preferablyc-kit^(pos) cardiac stem cells, which are cultured and expanded invitro.

In yet another embodiment, the present invention provides media for usein the culture and expansion of stem cells, preferably cardiac stemcells, more preferably c-kit^(pos) cardiac stem cells. Such media cancomprise DMEM/F12, patient serum, insulin, transferring and sodiumselenite. In one embodiment, the media can further comprise one or moreof human recombinant bFGF, human recombinant EGF, uridine and inosine.

In one embodiment, components of the medium can be present inapproximate ranges as follows:

Component Final Concentration Patient serum 5-20% by weight Humanrecombinant bFGF 10-100 ng/ml Human recombinant EGF 10-100 ng/ml Insulin2-20 μg/ml Transferrin 2-20 μg/ml Sodium selenite 2-10 ng/ml Uridine0.24-2.44 mg/ml Inosine 0.27-2.68 mg/ml

In anther embodiment, substitutions of the components of the media maybe made as known by those of skill in the art. For example, insulin canbe substituted with insulin-like growth factor I. Uridine and inosinecan be substituted with mixtures of other nucleotides, includingadenosine, guanosine, xanthine, thymidine, and cytidine.

In one embodiment of the present invention, the above media can beutilized during the culturing and expansion of stem cells that are to beadministered in order to regenerate or create new myocardium in adamaged or infarcted area of the heart.

In another embodiment of the invention, the cultured and expanded stemcells, preferably cardiac stem cells, more preferably c-kit^(pos)cardiac stem cells, are activated prior to their implantation ordelivery. In one embodiment, the stem cells are contacted with one ormore growth factors. Suitable growth factors can be any of thosedescribed herein, including, but not limited to: Activin A, AngiotensinII, Bone Morphogenic Protein 2, Bone Morphogenic Protein 4, BoneMorphogenic Protein 6, Cardiotrophin-1, Fibroblast Growth Factor 1,Fibroblast Growth Factor 4, Flt3 Ligand, Glial-Derived NeurotrophicFactor, Heparin, Hepatocyte Growth Factor, Insulin-like Growth Factor-I,Insulin-like Growth Factor-II, Insulin-Like Growth Factor BindingProtein-3, Insulin-Like Growth Factor Binding Protein-5, Interleukin-3,Interleukin-6, Interleukin-8, Leukemia Inhibitory Factor, Midkine,Platelet-Derived Growth Factor AA, Platelet-Derived Growth Factor BB,Progesterone, Putrescine, Stem Cell Factor, Stromal-Derived Factor-1,Thrombopoietin, Transforming Growth Factor-α, Transforming GrowthFactor-β1, Transforming Growth Factor-β2, Transforming Growth Factor-β3,Vascular Endothelial Growth Factor, Wnt1, Wnt3a, and Wnt5a, as describedin Ko, 2006; Kanemura, 2005; Kaplan, 2005; Xu, 2005; Quinn, 2005;Almeida, 2005; Barnabe-Heider, 2005; Madlambayan, 2005; Kamanga-Sollo,2005; Heese, 2005; He, 2005; Beattie, 2005; Sekiya, 2005; Weidt, 2004;Encabo, 2004; and Buytaeri-Hoefen, 2004, the entire text of each ofwhich is incorporated herein by reference. One of skill in the art willbe able to select one or more appropriate growth factors. In a preferredembodiment, the stem cells are contacted with hepatocyte growth factor(HGF) and/or insulin-like growth factor-1 (IGF-1). In one embodiment,the HGF is present in an amount of about 0-400 ng/ml. In a furtherembodiment, the HGF is present in an amount of about 25, about 50, about75, about 100, about 125, about 150, about 175, about 200, about 225,about 250, about 275, about 300, about 325, about 350, about 375 orabout 400 ng/ml. In another embodiment, the IGF-1 is present in anamount of about 0-500 ng/ml. In yet a further embodiment, the IGF-1 ispresent in an amount of about 25, about 50, about 75, about 100, about125, about 150, about 175, about 200, about 225, about 250, about 275,about 300, about 325, about 350, about 375, about 400, about 425, about450, about 475, or about 500 ng/ml.

In yet a still further embodiment, the one or more growth factors can bepresent in the media provided herein, such that in one embodiment, themedia comprises one or more growth factors, DMEM/F12, patient serum,insulin, transferring and sodium selenite and optionally one or more ofhuman recombinant bFGF, human recombinant EGF, uridine and inosine. Itis contemplated that the components of the media can be present in theamounts described herein, and one of skill in the art will be able todetermine a sufficient amount of the one or more growth factors in orderto obtain activation of any stem cells contacted therewith.

In one embodiment of the present invention, activated stem cells,preferably activated cardiac stem cells, more preferable activatedc-kit^(pos) cardiac stem cells are delivered to, or implanted in, anarea of the vasculature in need of therapy or repair. For example, inone embodiment the activated stem cells are delivered to, or implantedin, the site of an occluded or blocked cardiac vessel or artery. In oneembodiment of the present invention, cardiac stem cells that are c-kitpos and contain the flik-1 epitope are delivered to, or implanted in,the area in need of therapy or repair. In another embodiment of theinvention, the activated stem cells form into an artery or vessel at thesite at which the stem cells were delivered or implanted. In yet afurther embodiment, the formed artery or vessel has a diameter of over100 μm. In yet a further embodiment, the formed artery or vessel has adiameter of at least 125, at least 150, at least 175, at least 200, atleast 225, at least 250 or at least 275 μm. In yet another embodiment ofthe present invention, the formed artery or vessel provides a“biological bypass” around the area in need of therapy or repair,including around an occlusion or blockage such that blood flow, bloodpressure, and circulation are restored or improved. In yet a furtherstill embodiment of the present invention, the administration ofactivated stem cells can be done in conjunction with other therapeuticmeans, including but not limited to the administration of othertherapeutics, including one or more growth factors.

The pharmaceutical compositions of the present invention may be used astherapeutic agents—i.e. in therapy applications. As herein, the terms“treatment” and “therapy” include curative effects, alleviation effects,and prophylactic effects.

As used herein, “patient” may encompass any vertebrate including but notlimited to humans, mammals, reptiles, amphibians and fish. However,advantageously, the patient is a mammal such as a human, or an animalmammal such as a domesticated mammal, e.g., dog, cat, horse, and thelike, or production mammal, e.g., cow, sheep, pig, and the like.

As used herein “somatic stem cell” or “stem cell” or “hematopoieticcell” refers to either autologous or allogenic stem cells, which may beobtained from the bone marrow, peripheral blood, or other source.

As used herein, “adult” stem cells refers to stem cells that are notembryonic in origin nor derived from embryos or fetal tissue.

As used herein “recently damaged myocardium” refers to myocardium whichhas been damaged within one week of treatment being started. In apreferred embodiment, the myocardium has been damaged within three daysof the start of treatment. In a further preferred embodiment, themyocardium has been damaged within 12 hours of the start of treatment.It is advantageous to employ stem cells alone or in combination withcytokine(s) as herein disclosed to a recently damaged myocardium.

As used herein “damaged myocardium” refers to myocardial cells whichhave been exposed to ischemic conditions. These ischemic conditions maybe caused by a myocardial infarction, or other cardiovascular disease orrelated complaint. The lack of oxygen causes the death of the cells inthe surrounding area, leaving an infarct, which will eventually scar.

As used herein, “home” refers to the attraction and mobilization ofsomatic stem cells towards damaged myocardium and/or myocardial cells.

As used herein, “assemble” refers to the assembly of differentiatedsomatic stem cells into functional structures i.e., myocardium and/ormyocardial cells, coronary arteries, arterioles, and capillaries etc.This assembly provides functionality to the differentiated myocardiumand/or myocardial cells, coronary arteries, arterioles and capillaries.

Thus, the invention involves the use of somatic stem cells. These arepresent in animals in small amounts, but methods of collecting stemcells are known to those skilled in the art.

In another aspect of the invention, the stem cells are selected to belineage negative. The term “lineage negative” is known to one skilled inthe art as meaning the cell does not express antigens characteristic ofspecific cell lineages.

Advantageously, the lineage negative stem cells are selected to be c-kitpositive. The term “c-kit” is known to one skilled in the art as being areceptor which is known to be present on the surface of stem cells, andwhich is routinely utilized in the process of identifying and separatingstem cells from other surrounding cells.

The invention further involves a therapeutically effective dose oramount of stem cells applied to the heart. An effective dose is anamount sufficient to effect a beneficial or desired clinical result.Said dose could be administered in one or more administrations. In theexamples that follow, 2×10⁴-1×10⁵ stem cells were administered in themouse model. While there would be an obvious size difference between thehearts of a mouse and a human, it is possible that this range of stemcells would be sufficient in a human as well. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including their size, age, sizeof the infarct, and amount of time since damage. One skilled in the art,specifically a physician or cardiologist, would be able to determine thenumber of stem cells that would constitute an effective dose withoutundue experimentation.

In another aspect of the invention, the stem cells are delivered to theheart, specifically to the border area of the infarct. As one skilled inthe art would be aware, the infarcted area is visible grossly, allowingthis specific placement of stem cells to be possible.

The stem cells are advantageously administered by injection,specifically an intramyocardial injection. As one skilled in the artwould be aware, this is the preferred method of delivery for stem cellsas the heart is a functioning muscle. Injection of the stem cells intothe heart ensures that they will not be lost due to the contractingmovements of the heart.

In a further aspect of the invention, the stem cells are administered byinjection transendocardially or trans-epicardially. This preferredembodiment allows the stem cells to penetrate the protective surroundingmembrane, necessitated by the embodiment in which the cells are injectedintramyocardially.

A preferred embodiment of the invention includes use of a catheter-basedapproach to deliver the trans-endocardial injection. The use of acatheter precludes more invasive methods of delivery wherein the openingof the chest cavity would be necessitated. As one skilled in the art isaware, optimum time of recovery would be allowed by the more minimallyinvasive procedure, which as outlined here, includes a catheterapproach.

A catheter approach includes the use of such techniques as the NOGAcatheter or similar systems. The NOGA catheter system facilitates guidedadministration by providing electromechanic mapping of the area ofinterest, as well as a retractable needle that can be used to delivertargeted injections or to bathe a targeted area with a therapeutic. Anyof the embodiments of the present invention can be administered throughthe use of such a system to deliver injections or provide a therapeutic.One of skill in the art will recognize alternate systems that alsoprovide the ability to provide targeted treatment through theintegration of imaging and a catheter delivery system that can be usedwith the present invention. Information regarding the use of NOGA andsimilar systems can be found in, for example, Sherman, 2003; Patel,2005; and Perrin, 2003; the text of each of which are incorporatedherein in their entirety.

Further embodiments of the invention require the stem cells to migrateinto the infarcted region and differentiate into myocytes, smooth musclecells, and endothelial cells. It is known in the art that these types ofcells must be present to restore both structural and functionalintegrity. Other approaches to repairing infarcted or ischemic tissuehave involved the implantation of these cells directly into the heart,or as cultured grafts, such as in U.S. Pat. Nos. 6,110,459, and6,099,832.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare essential for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. The ability to restoreboth functional and structural integrity is yet another aspect of thisinvention.

Another aspect of the invention relates to the administration of acytokine. This cytokine may be chosen from a group of cytokines, or mayinclude combinations of cytokines. Stem cell factor (SCF) andgranulocyte-colony stimulating factor (G-CSF) are known by those skilledin the art as stimulating factors which cause the mobilization of stemcells into the blood stream (Bianco et al, 2001, Clutterbuck, 1997,Kronenwett et al, 2000, Laluppa et al, 1997, Patchen et al, 1998).Stromal cell-derived factor-1 has been shown to stimulate stem cellmobilization chemotactically, while steel factor has both chemotacticand chemokinetic properties (Caceres-Cortes et al, 2001, Jo et al, 2000,Kim and Broxmeyer, 1998, Ikuta et al, 1991). Vascular endothelial growthfactor has been surmised to engage a paracrine loop that helpsfacilitate migration during mobilization (Bautz et al, 2000,Janowska-Wieczorek et al, 2001). Macrophage colony stimulating factorand granulocyte-macrophage stimulating factor have been shown tofunction in the same manner of SCF and G-CSF, by stimulatingmobilization of stem cells. Interleukin-3 has also been shown tostimulate mobilization of stem cells, and is especially potent incombination with other cytokines.

The cytokine can be administered via a vector that expresses thecytokine in vivo. A vector for in vivo expression can be a vector orcells or an expression system as cited in any document incorporatedherein by reference or used in the art, such as a viral vector, e.g., anadenovirus, poxvirus (such as vaccinia, canarypox virus, MVA, NYVAC,ALVAC, and the like), lentivirus or a DNA plasmid vector; and, thecytokine can also be from in vitro expression via such a vector or cellsor expression system or others such as a baculovirus expression system,bacterial vectors such as E. coli, and mammalian cells such as CHOcells. See, e.g., U.S. Pat. Nos. 6,265,189, 6,130,066, 6,004,777,5,990,091, 5,942,235, 5,833,975. The cytokine compositions may lendthemselves to administration by routes outside of those stated to beadvantageous or preferred for stem cell preparations; but, cytokinecompositions may also be advantageously administered by routes stated tobe advantageous or preferred for stem cell preparations.

A further aspect of the invention involves administration of atherapeutically effective dose or amount of a cytokine. An effectivedose is an amount sufficient to effect a beneficial or desired clinicalresult. Said dose could be administered in one or more administrations.In a preferred embodiment, the dose would be given over the course ofabout two or three days following the beginning of treatment. However,the precise determination of what would be considered an effective dosemay be based on factors individual to each patient, including theirsize, age, size of the infarct, the cytokine or combination of cytokinesbeing administered, and amount of time since damage. One skilled in theart, specifically a physician or cardiologist, would be able todetermine a sufficient amount of cytokine that would constitute aneffective dose without being subjected to undue experimentation.

The invention also involves the administration of the therapeuticallyeffective dose or amount of a cytokine being delivered by injection,specifically subcutaneously or intravenously. A person skilled in theart will be aware that subcutaneous injection or intravenous deliveryare extremely common and offer an effective method of delivering thespecific dose in a manner which allows for timely uptake and circulationin the blood stream.

A further aspect of the invention includes the administered cytokinestimulating the patient's stem cells and causing mobilization into theblood stream. As mentioned previously, the given cytokines arewell-known to one skilled in the art for their ability to promote saidmobilization.

Advantageously, once the stem cells have mobilized into the bloodstream,they home to the damaged area of the heart, as will become clear throughthe following examples.

Further embodiments of the invention involve the stem cells migratinginto the infarcted region and differentiating into myocytes, smoothmuscle cells, and endothelial cells. It is known in the art that thesetypes of cells must be present to restore both structural and functionalintegrity.

A further embodiment of the invention includes administering aneffective amount of one or more cytokines to the infarcted region. Aneffective dose is an amount sufficient to effect a beneficial or desiredclinical result. Said dose could be administered in one or moreadministrations. However, the precise determination of what would beconsidered an effective dose may be based on factors individual to eachpatient, including their size, age, size of the infarct, the cytokine orcombination of cytokines being administered, and amount of time sincedamage. One skilled in the art, specifically a physician orcardiologist, would be able to determine a sufficient amount of cytokinethat would constitute an effective dose without being subjected to undueexperimentation.

A still further embodiment of the invention includes the administeringof an effective amount of one or more cytokines to the heart byinjection. Preferably, the cytokines are delivered to the infarctedregion or to the area bordering the infarcted region. As one skilled inthe art would be aware, the infarcted area is visible grossly, allowingthis specific placement of cytokines to be possible.

The cytokines are advantageously administered by injection, specificallyan intramyocardial injection. As one skilled in the art would be aware,this is the preferred method of delivery for cytokines as the heart is afunctioning muscle. Injection of the cytokines into the heart ensuresthat they will not be lost due to the contracting movements of theheart.

In a further aspect of the invention, the cytokines are administered byinjection transendocardially or trans-epicardially. This preferredembodiment allows the cytokines to penetrate the protective surroundingmembrane, necessitated by the embodiment in which the cytokines areinjected intramyocardially.

A preferred embodiment of the invention includes use of a catheter-basedapproach to deliver the trans-endocardial injection. The use of acatheter precludes more invasive methods of delivery wherein the openingof the chest cavity would be necessitated. As one skilled in the art isaware, optimum time of recovery would be allowed by the more minimallyinvasive procedure, which as outlined here, includes a catheterapproach.

A further embodiment of the invention includes the delivery of thecytokines by a single administration. A still further embodiment of theinvention includes multiple administrations of the same dosage ofcytokines to the heart. A still further embodiment of the inventionincludes administration of multiple doses of the cytokines to the heart,such that a gradient is formed.

A still further embodiment of the invention includes the stimulation,migration, proliferation and/or differentiation of the resident cardiacstem cells.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare important for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. The ability to restoreboth functional and structural integrity or better functional andstructural integrity than previously achieved in the art is yet anotheraspect of this invention.

It is a preferred in the practice of the invention to utilize both theadministration of stem cells and that of a cytokine to ensure the mosteffective method of repairing damaged myocardium.

Stem cells employed in the invention are advantageously selected to belineage negative. The term “lineage negative” is known to one skilled inthe art as meaning the cell does not express antigens characteristic ofspecific cell lineages. And, it is advantageous that the lineagenegative stem cells are selected to be c-kit positive. The term “c-kit”is known to one skilled in the art as being a receptor which is known tobe present on the surface of stem cells, and which is routinely utilizedin the process of identifying and separating stem cells from othersurrounding cells.

In certain embodiments, a therapeutically effective dose of stem cellsis applied, delivered, or administered to the heart or implanted intothe heart. An effective dose or amount is an amount sufficient to effecta beneficial or desired clinical result. Said dose could be administeredin one or more administrations. In the examples that follow, 2×10⁴-1×10⁵stem cells were administered in the mouse model. While there would be anobvious size difference between the hearts of a mouse and a human, it ispossible that 2×10⁴-1×10⁵ stem cells would be sufficient in a human aswell. However, the precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, size of the infarct, and amount of time sincedamage. One skilled in the art, specifically a physician orcardiologist, would be able to determine the number and type (or types)of stem cells which would constitute an effective dose without beingsubjected to undue experimentation, from this disclosure and theknowledge in the art; and, in this regard and in general in regard topreparing formulations and administering formulations or componentsthereof, mention is made of the teachings in the Examples and that theskilled artisan can scale dosages, amounts and the like based on theweight of the patient to be treated in comparison to the weight of anyanimal employed in the Examples. The stem cells are advantageously bonemarrow or are cardiac stem cells; and even more advantageously, the stemcells are adult bone marrow (hematopoietic stem cells) or adult cardiacstem cells or a combination thereof or a combination of cardiac stemcells such as adult cardiac stem cells and another type of stem cellsuch as another type of adult stem cells.

In another aspect of the invention, the stem cells are delivered to theheart, specifically to the border area of the infarct. As one skilled inthe art would be aware, the infarcted area is visible grossly, allowingthis specific placement of stem cells to be possible.

The stem cells are advantageously administered by injection,specifically an intramyocardial injection. As one skilled in the artwould be aware, this is the preferred method of delivery for stem cellsas the heart is a functioning muscle. Injection of the stem cells intothe heart ensures that they will not be lost due to the contractingmovements of the heart.

In other aspects of the invention, the stem cells are administered byinjection transendocardially or trans-epicardially. This preferredembodiment allows the stem cells to penetrate the protective surroundingmembrane, necessitated by the embodiment in which the cells are injectedintramyocardially.

A preferred embodiment of the invention includes use of a catheter-basedapproach to deliver the trans-endocardial injection. The use of acatheter precludes more invasive methods of delivery wherein the openingof the chest cavity would be necessitated. As one skilled in the art isaware, optimum time of recovery would be allowed by the more minimallyinvasive procedure, which as outlined here, includes a catheterapproach.

Embodiments of the invention can involve the administration of acytokine. This cytokine may be chosen from a group of cytokines, or mayinclude combinations of cytokines.

A further aspect of the invention involves administration of atherapeutically effective dose of a cytokine. An effective dose oramount is an amount sufficient to effect a beneficial or desiredclinical result. Said dose could be administered in one or moreadministrations. In a preferred embodiment, the dose would be given overthe course of about two or three days following the beginning oftreatment. However, the precise determination of what would beconsidered an effective dose may be based on factors individual to eachpatient, including their size, age, size of the infarct, the cytokine orcombination of cytokines being administered, and amount of time sincedamage. One skilled in the art, specifically a physician orcardiologist, would be able to determine a sufficient amount of cytokinethat would constitute an effective dose without being subjected to undueexperimentation, especially in view of the disclosure herein and theknowledge in the art.

The administration of the therapeutically effective dose of at least onecytokine is advantageously by injection, specifically subcutaneously orintravenously. A person skilled in the art will be aware thatsubcutaneous injection or intravenous delivery are extremely common andoffer an effective method of delivering the specific dose in a mannerwhich allows for timely uptake and circulation in the blood stream.

A further aspect of the invention includes the administered cytokinestimulating the patient's stem cells and causing mobilization into theblood stream. As mentioned previously, the given cytokines are wellknown to one skilled in the art for their ability to promote saidmobilization. Again, once the stem cells have mobilized into thebloodstream, they home to the damaged area of the heart. Thus in certainembodiments, both the implanted stem cells and the mobilized stem cellsmigrate into the infarct region and differentiate into myocytes, smoothmuscle cells, and endothelial cells. It is known in the art that thesetypes of cells are advantageously present to restore both structural andfunctional integrity.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare essential for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. Cardiac structures canbe generated ex vivo and then implanted in the form of a graft; with theimplantation of the graft being alone or in combination with stem cellsor stem cells and at least one cytokine as in this disclosure, e.g.,advantageously adult or cardiac or hematopoietic stem cells such asadult cardiac and/or adult hematopoietic stem cells or adult cardiacstem cells with another type of stem cell e.g. another type of adultstem cell. The means of generating and/or regenerating myocardium exvivo, may incorporate somatic stem cells and heart tissue being culturedin vitro, optionally in the presence of a cytokine. The somatic stemcells differentiate into myocytes, smooth muscle cells and endothelialcells, and proliferate in vitro, forming myocardial tissue and/or cells.These tissues and cells may assemble into cardiac structures includingarteries, arterioles, capillaries, and myocardium. The tissue and/orcells formed in vitro may then be implanted into a patient, e.g. via agraft, to restore structural and functional integrity.

Additionally or alternatively, the source of the tissue being graftedcan be from other sources of tissue used in grafts of the heart.

The restoration or some restoration of both functional and structuralintegrity of cardiac tissue-advantageously over that which has occurredpreviously—is yet another aspect of this invention.

Accordingly, the invention comprehends, in further aspects, methods forpreparing compositions such as pharmaceutical compositions includingsomatic stem cells and/or at least one cytokine, for instance, for usein inventive methods for treating cardiovascular disease or conditionsor cardiac conditions.

The present invention is additionally described by way of the following,non-limiting examples, that provide a better understanding of thepresent invention and of its many advantages.

All of the materials, reagents, chemicals, assays, cytokines,antibodies, and miscellaneous items referred to in the followingexamples are readily available to the research community throughcommercial suppliers, including but not limited to, Genzyme, Invitrogen,Gibco BRL, Clonetics, Fisher Scientific, R & D Systems, MBLInternational Corporation, CN Biosciences Corporate, Sigma Aldrich, andCedarLane Laboratories, Limited.

For example,

-   -   stem cell factor is available under the name SCF (multiple forms        of recombinant human, recombinant mouse, and antibodies to        each), from R & D Systems (614 McKinley Place N.E., Minneapolis,        Minn. 55413);    -   granulocyte-colony stimulating factor is available under the        name G-CSF (multiple forms of recombinant human, recombinant        mouse, and antibodies to each), from R & D Systems;    -   stem cell antibody-1 is available under the name SCA-1 from MBL        International Corporation (200 Dexter Avenue, Suite D,        Watertown, Mass. 02472);    -   multidrug resistant antibody is available under the name        Anti-MDR from CN Biosciences Corporate;    -   c-kit antibody is available under the name c-kit (Ab-1)        Polyclonal Antibody from CN Biosciences Corporate (Affiliate of        Merck KgaA, Darmstadt, Germany. Corporate headquarters located        at 10394 Pacific Center Court, San Diego, Calif. 92121).

EXAMPLES Example 1 Hematopoietic Stem Cell (HSC) Repair of InfarctedMyocardium

A. Harvesting of Hematopoietic Stem Cells

Bone marrow was harvested from the femurs and tibias of male transgenicmice expressing enhanced green fluorescent protein (EGFP). Aftersurgical removal of the femurs and tibias, the muscle was dissected andthe upper and lower surface of the bone was cut on the surface to allowthe collecting buffer to infiltrate the bone marrow. The fluidcontaining buffer and cells was collected in tubes such as 1.5 mlEpindorf tubes. Bone marrow cells were suspended in PBS containing 5%fetal calf serum (FCS) and incubated on ice with rat anti-mousemonoclonal antibodies specific for the following hematopoietic lineages:CD4 and CD8 (T-lymphocytes), B-220 (B-lymphocytes), Mac-1 (macrophages),GR-1 (granulocytes) (Caltag Laboratories) and TER-119 (erythrocytes)(Pharmingen). Cells were then rinsed in PBS and incubated for 30 minuteswith magnetic beads coated with goat anti-rat immunoglobulin(Polysciences Inc.). Lineage positive cells (Lin⁺) were removed by abiomagnet and lineage negative cells (Lin⁻) were stained withACK-4-biotin (anti-c-kit mAb). Cells were rinsed in PBS, stained withstreptavidin-conjugated phycoerythrin (SA-PE) (Caltag Labs.) and sortedby fluorescence activated cell sorting (FACS) using a FACSVantageinstrument (Becton Dickinson). Excitation of EGFP and ACK-4-biotin-SA-EPoccurred at a wavelength of 488 nm. The Lin⁻ cells were sorted as c-kitpositive (c-kit^(pos)) and c-kit negative (c-kit^(NEG)) with a 1-2 logdifference in staining intensity (FIG. 1). The c-kit^(POS) cells weresuspended at 2×10⁴ to 1×10⁵ cells in 5 μl of PBS and the c-kit^(NEG)cells were suspended at a concentration of 1×10⁵ in 5 μl of PBS.

B. Induction of Myocardial Infarction in Mice

Myocardial infarction was induced in female C57BL/6 mice at 2 months ofage as described by Li et al. (1997). Three to five hours afterinfarction, the thorax of the mice was reopened and 2.5 μl of PBScontaining Lin⁻ c-kit^(POS) cells were injected in the anterior andposterior aspects of the viable myocardium bordering the infarct (FIG.2). Infarcted mice, left uninjected or injected with Lin⁻ c-kit^(NEG)cells, and sham-operated mice i.e., mice where the chest cavity wasopened but no infarction was induced, were used as controls. All animalswere sacrificed 9±2 days after surgery. Protocols were approved byinstitutional review board. Results are presented as mean±SD.Significance between two measurements was determined by the Student's ttest, and in multiple comparisons was evaluated by the Bonferroni method(Scholzen and Gerdes, 2000). P<0.05 was considered significant.

Injection of male Lin⁻ c-kit^(POS) bone marrow cells in theperi-infarcted left ventricle of female mice resulted in myocardialregeneration. The peri-infarcted region is the region of viablemyocardium bordering the infarct. Repair was obtained in 12 of 30 mice(40%). Failure to reconstitute infarcts was attributed to the difficultyof transplanting cells into tissue contracting at 600 beats per minute(bpm). However, an immunologic reaction to the histocompatibilityantigen on the Y chromosome of the donor bone marrow cells could accountfor the lack of repair in some of the female recipients. Closely packedmyocytes occupied 68±11% of the infarcted region and extended from theanterior to the posterior aspect of the ventricle (FIGS. 2A-2D). Newmyocytes were not found in mice injected with Lin⁻ c-kit^(NEG) cells(FIG. 2E).

C. Determination of Ventricular Function

Mice were anesthetized with chloral hydrate (400 mg/kg body weight,i.p.), and the right carotid artery was cannulated with a microtippressure transducer (model SPR-671, Millar) for the measurements of leftventricular (LV) pressures and LV+ and −dP/dt in the closed-chestpreparation to determine whether developing myocytes derived from theHSC transplant had an impact on function. Infarcted mice non-injected orinjected with Lin⁻ c-kit^(NEG) cells were combined in the statistics. Incomparison with sham-operated groups, the infarcted groups exhibitedindices of cardiac failure (FIG. 3). In mice treated with Lin⁻c-kit^(POS) cells, LV end-diastolic pressure (LVEDP) was 36% lower, anddeveloped pressure (LVDP) and LV+ and −dP/dt were 32%, 40%, and 41%higher, respectively (FIG. 4A).

D. Determination of Cell Proliferation and EGFP Detection

The abdominal aorta was cannulated, the heart was arrested in diastoleby injection of cadmium chloride (CdCl₂), and the myocardium wasperfused retrogradely with 10% buffered formalin. Three tissue sections,from the base to the apex of the left ventricle, were stained withhematoxylin and eosin. At 9±2 days after coronary occlusion, theinfarcted portion of the ventricle was easily identifiable grossly andhistologically (see FIG. 2A). The lengths of the endocardial andepicardial surfaces delimiting the infarcted region, and the endocardiumand epicardium of the entire left ventricle were measured in eachsection. Subsequently, their quotients were computed to yield theaverage infarct size in each case. This was accomplished at 4×magnification utilizing an image analyzer connected to a microscope. Thefraction of endocardial and epicardial circumference delimiting theinfarcted area (Pfeffer and Braunwald, 1990; Li et al., 1997) did notdiffer in untreated mice, 78±18% (n=8) and in mice treated with Lin⁻c-kit^(POS) cells (n=12), 75±14% or Lin⁻ c-kit^(NEG) cells (n=11),75±15%.

To establish whether Lin⁻ c-kit^(POS) cells resulted in myocardialregeneration, BrdU (50 mg/kg body weight, i.p.) was administered dailyto the animals for 4-5 consecutive days before sacrifice to determinecumulative cell division during active growth. Sections were incubatedwith anti-BrdU antibody and BrdU labeling of cardiac cell nuclei in theS phase was measured. Moreover, expression of Ki67 in nuclei (Ki67 isexpressed in cycling cells in G1, S, G2, and early mitosis) wasevaluated by treating samples with a rabbit polyclonal anti-mouse Ki67antibody (Dako Corp.). FITC-conjugated goat anti-rabbit IgG was used assecondary antibody. (FIGS. 5 and 6). EGFP was detected with a rabbitpolyclonal anti-GFP (Molecular Probes). Myocytes were recognized with amouse monoclonal anti-cardiac myosin heavy chain (MAB 1548; Chemicon) ora mouse monoclonal anti-α-sarcomeric actin (clone 5C5; Sigma),endothelial cells with a rabbit polyclonal anti-human factor VIII(Sigma) and smooth muscle cells with a mouse monoclonal anti-α-smoothmuscle actin (clone 1A4; Sigma). Nuclei were stained with propidiumiodide (PI), 10 μg/ml. The percentages of myocyte (M), endothelial cell(EC) and smooth muscle cell (SMC) nuclei labeled by BrdU and Ki67 wereobtained by confocal microscopy. This was accomplished by dividing thenumber of nuclei labeled by the total number of nuclei examined. Numberof nuclei sampled in each cell population was as follows; BrdU labeling:M=2,908; EC=2,153; SMC=4,877. Ki67 labeling: M=3,771; EC=4,051;SMC=4,752. Number of cells counted for EGFP labeling: M=3,278; EC=2,056;SMC=1,274. The percentage of myocytes in the regenerating myocardium wasdetermined by delineating the area occupied by cardiac myosin stainedcells divided by the total area represented by the infarcted region ineach case. Myocyte proliferation was 93% (p<0.001) and 60% (p<0.001)higher than in endothelial cells, and 225% (p<0.001 and 176% (p<0.001)higher than smooth muscle cells, when measured by BrdU and Ki67,respectively.

The origin of the cells in the forming myocardium was determined by theexpression of EGFP (FIGS. 7 and 8). EGFP expression was restricted tothe cytoplasm and the Y chromosome to nuclei of new cardiac cells. EGFPwas combined with labeling of proteins specific for myocytes,endothelial cells and smooth muscle cells. This allowed theidentification of each cardiac cell type and the recognition ofendothelial cells and smooth muscle cells organized in coronary vessels(FIGS. 5, 7, and 8). The percentage of new myocytes, endothelial cellsand smooth muscle cells that expressed EGFP was 53±9% (n=7), 44±6% (n=7)and 49±7% (n=7), respectively. These values were consistent with thefraction of transplanted Lin⁻ c-kit^(POS) bone marrow cells thatexpressed EGFP, 44±10% (n=6). An average 54±8% (n=6) of myocytes,endothelial cells and smooth muscle cells expressed EGFP in the heart ofdonor transgenic mice.

E. Detection of the Y-Chromosome

For the fluorescence in situ hybridization (FISH) assay, sections wereexposed to a denaturing solution containing 70% formamide. Afterdehydration with ethanol, sections were hybridized with the DNA probeCEP Y (satellite III) Spectrum Green (Vysis) for 3 hours. Nuclei werestained with PI.

Y-chromosomes were not detected in cells from the surviving portion ofthe ventricle. However, the Y-chromosome was detected in the newlyformed myocytes, indicating their origin as from the injected bonemarrow cells (FIG. 9).

F. Detection of Transcription Factors and Connexin 43

Sections were incubated with rabbit polyclonal anti-MEF2 (C-21; SantaCruz), rabbit polyclonal anti-GATA-4 (H-112; Santa Cruz), rabbitpolyclonal anti-Csx/NRx2.5 (obtained from Dr. Izumo) and rabbitpolyclonal anti-connexin 43 (Sigma). FITC-conjugated goat anti-rabbitIgG (Sigma) was used as secondary antibody.

To confirm that newly formed myocytes represented maturing cells aimingat functional competence, the expression of the myocyte enhancer factor2 (MEF2), the cardiac specific transcription factor GATA-4 and the earlymarker of myocyte development Csx/NRx2.5 was examined. In the heart,MEF2 proteins are recruited by GATA-4 to synergistically activate thepromoters of several cardiac genes such as myosin light chain, troponinT, troponin I, α-myosin heavy chain, desmin, atrial natriuretic factorand α-actin (Durocher et al., 1997; Morin et al., 2000). Csx/NRx2.5 is atranscription factor restricted to the initial phases of myocytedifferentiation (Durocher et al., 1997). In the reconstituting heart,all nuclei of cardiac myosin labeled cells expressed MEF2 (FIGS. 7D-7F)and GATA-4 (FIG. 10), but only 40±9% expressed Csx/NRx2.5 (FIGS. 7G-7I).To characterize further the properties of these myocytes, the expressionof connexin 43 was determined. This protein is responsible forintercellular connections and electrical coupling through the generationof plasma membrane channels between myocytes (Beardsle et al., 1998;Musil et al., 2000); connexin 43 was apparent in the cell cytoplasm andat the surface of closely aligned differentiating cells (FIGS. 11A-11D).These results were consistent with the expected functional competence ofthe heart muscle phenotype. Additionally, myocytes at various stages ofmaturation were detected within the same and different bands (FIG. 12).

Example 2 Mobilization of Bone Marrow Cells to Repair InfarctedMyocardium

A. Myocardial Infarction and Cytokines.

Fifteen C57BL/6 male mice at 2 months of age were splenectomized and 2weeks later were injected subcutaneously with recombinant rat stem cellfactor (SCF), 200 μg/kg/day, and recombinant human granulocyte colonystimulating factor (G-CSF), 50 μg/kg/day (Amgen), once a day for 5 days(Bodine et al., 1994; Orlic et al., 1993). Under ether anesthesia, theleft ventricle (LV) was exposed and the coronary artery was ligated(Orlic et al., 2001; Li et al., 1997; Li et al., 1999). SCF and G-CSFwere given for 3 more days. Controls consisted of splenectomizedinfarcted and sham-operated (SO) mice injected with saline. BrdU, 50mg/kg body weight, was given once a day, for 13 days, before sacrifice;mice were killed at 27 days. Protocols were approved by New York MedicalCollege. Results are mean±SD. Significance was determined by theStudent's t test and Bonferroni method (Li et al., 1999). Mortality wascomputed with log-rank test. P<0.05 was significant.

Given the ability of bone marrow Lin⁻ c-kit^(POS) cells totransdifferentiate into the cardiogenic lineage (Orlic et al., 2001), aprotocol was used to maximize their number in the peripheral circulationin order to increase the probability of their homing to the region ofdead myocardium. In normal animals, the frequency of Lin⁻ c-kit^(POS)cells in the blood is only a small fraction of similar cells present inthe bone marrow (Bodine et al., 1994; Orlic et al., 1993). As documentedpreviously, the cytokine treatment used here promotes a marked increaseof Lin⁻ c-kit^(POS) cells in the bone marrow and a redistribution ofthese cells from the bone marrow to the peripheral blood. This protocolleads to a 250-fold increase in Lin⁻ c-kit^(POS) cells in thecirculation (Bodine et al., 1994; Orlic et al., 1993).

In the current study, BMC mobilization by SCF and G-CSF resulted in adramatic increase in survival of infarcted mice; with cytokinetreatment, 73% of mice (11 of 15) survived 27 days, while mortality wasvery high in untreated infarcted mice (FIG. 13A). A large number ofanimals in this group died from 3 to 6 days after myocardial infarction(MI) and only 17% (9 of 52) reached 27 days (p<0.001). Mice that diedwithin 48 hours post-MI were not included in the mortality curve tominimize the influence of the surgical trauma. Infarct size was similarin the cytokine-, 64±11% (n=11), and saline-, 62±9% (n=9), injectedanimals as measured by the number of myocytes lost in the leftventricular free wall (LVFW) at 27 days (FIG. 14).

Importantly, bone marrow cell mobilization promoted myocardialregeneration in all 11 cytokine-treated infarcted mice, sacrificed 27days after surgery (FIG. 13B). Myocardial growth within the infarct wasalso seen in the 4 mice that died prematurely at day 6 (n=2) and at day9 (n=2). Cardiac repair was characterized by a band of newly formedmyocardium occupying most of the damaged area. The developing tissueextended from the border zone to the inside of the injured region andfrom the endocardium to the epicardium of the LVFW. In the absence ofcytokines, myocardial replacement was never observed and healing withscar formation was apparent (FIG. 13C). Conversely, only small areas ofcollagen accumulation were detected in treated mice.

B. Detection of BMC Mobilization by Echocardiography and Hemodynamics.

Echocardiography was performed in conscious mice using a Sequoia 256c(Acuson) equipped with a 13-MHz linear transducer (15L8). The anteriorchest area was shaved and two dimensional (2D) images and M-modetracings were recorded from the parasternal short axis view at the levelof papillary muscles. From M-mode tracings, anatomical parameters indiastole and systole were obtained (Pollick et al., 1995). Ejectionfraction (EF) was derived from LV cross sectional area in 2D short axisview (Pollick et al., 1995): EF=[(LVDA−LVSA)/LVDA]*100 where LVDA andLVSA correspond to LV areas in diastole and in systole. Mice wereanesthetized with chloral hydrate (400 mg/kg body weight, ip) and amicrotip pressure transducer (SPR-671, Millar) connected to a chartrecorder was advanced into the LV for the evaluation of pressures and +and −dP/dt in the closed-chest preparation (Orlic et al., 2001; Li etal., 1997; Li et al., 1999).

EF was 48%, 62% and 114% higher in treated than in non-treated mice at9, 16 and 26 days after coronary occlusion, respectively (FIG. 15D). Inmice exposed to cytokines, contractile function developed with time inthe infarcted region of the wall (FIG. 15E-M; FIG. 16H-P, www.pnas.org).Conversely, LV end-diastolic pressure (LVEDP) increased 76% more innon-treated mice. The changes in LV systolic pressure (not shown),developed pressure (LVDP), + and −dP/dt were also more severe in theabsence of cytokine treatment (FIG. 17A-D). Additionally, the increasein diastolic stress in the zone bordering and remote from infarction was69-73% lower in cytokine-treated mice (FIG. 15N). Therefore,cytokine-mediated infarct repair restored a noticeable level ofcontraction in the regenerating myocardium, decreasing diastolic wallstress and increasing ventricular performance. Myocardial regenerationattenuated cavitary dilation and mural thinning during the evolution ofthe infarcted heart in vivo.

Echocardiographically, LV end-systolic (LVESD) and end-diastolic (LVEDD)diameters increased more in non-treated than in cytokine-treated mice,at 9, 16 and 26 days after infarction (FIG. 16A-B). Infarction preventedthe evaluation of systolic (AWST) and diastolic (AWDT) anterior wallthickness. When measurable, the posterior wall thickness in systole(PWST) and diastole (PWDT) was greater in treated mice (FIG. 16C-D).Anatomically, the wall bordering and remote from infarction was 26% and22% thicker in cytokine-injected mice (FIG. 16E). BMC-induced repairresulted in a 42% higher wall thickness-to-chamber radius ratio (FIG.15A). Additionally, tissue regeneration decreased the expansion incavitary diameter, −14%, longitudinal axis, −5% (FIG. 16F-G), andchamber volume, −26% (FIG. 15B). Importantly, ventricularmass-to-chamber volume ratio was 36% higher in treated animals (FIG.15C). Therefore, BMC mobilization that led to proliferation anddifferentiation of a new population of myocytes and vascular structuresattenuated the anatomical variables which define cardiac decompensation.

C. Cardiac Anatomy and Determination of Infarct Size.

Following hemodynamic measurements, the abdominal aorta was cannulated,the heart was arrested in diastole with CdCl₂ and the myocardium wasperfused with 10% formalin. The LV chamber was filled with fixative at apressure equal to the in vivo measured end-diastolic pressure (Li etal., 1997; Li et al., 1999). The LV intracavitary axis was measured andthree transverse slices from the base, mid-region and apex were embeddedin paraffin. The mid-section was used to measure LV thickness, chamberdiameter and volume (Li et al., 1997; Li et al., 1999). Infarct size wasdetermined by the number of myocytes lost from the LVFW (Olivetti etal., 1991; Beltrami et al., 1994).

To quantify the contribution of the developing band to the ventricularmass, firstly the volume of the LVFW (weight divided by 1.06 g/ml) wasdetermined in each group of mice. The data was 56±2 mm³ in sham operated(SO), 62±4 mm³ (viable FW=41±3; infarcted FW=21±4) in infarctednon-treated animals, and 56±9 mm³ (viable FW=37±8; infarcted FW=19±5) ininfarcted cytokine-treated mice. These values were compared to theexpected values of spared and lost myocardium at 27 days, given the sizeof the infarct in the non-treated and cytokine-treated animals. From thevolume of the LVFW (56 mm³) in SO and infarct size in non-treated, 62%,and treated, 64%, mice, it was possible to calculate the volume ofmyocardium destined to remain (non-treated=21 mm³; treated=20 mm³) anddestined to be lost (non-treated=35 mm³; treated=36 mm³) 27 days aftercoronary occlusion (FIG. 18A). The volume of newly formed myocardium wasdetected exclusively in cytokine-treated mice and found to be 14 mm³(FIG. 18A). Thus, the repair band reduced infarct size from 64% (36mm³/56 mm³=64%) to 39% [(36 mm³-14 mm³)/56 mm³=39%]. Since the sparedportion of the LVFW at 27 days was 41 and 37 mm³ in non-treated andtreated mice (see above), the remaining myocardium, shown in FIG. 18 a,underwent 95% (p<0.001) and 85% (p<0.001) hypertrophy, respectively.Consistently, myocyte cell volume increased 94% and 77% (FIG. 18B).

D. Determination the Total Volume of Formed Myocardium

The volume of regenerating myocardium was determined by measuring ineach of three sections the area occupied by the restored tissue andsection thickness. The product of these two variables yielded the volumeof tissue repair in each section. Values in the three sections wereadded and the total volume of formed myocardium was obtained.Additionally, the volume of 400 myocytes was measured in each heart.Sections were stained with desmin and laminin antibodies and propidiumiodide (PI). Only longitudinally oriented cells with centrally locatednuclei were included. The length and diameter across the nucleus werecollected in each myocyte to compute cell volume, assuming a cylindricalshape (Olivetti et al., 1991; Beltrami et al., 1994). Myocytes weredivided in classes and the number of myocytes in each class wascalculated from the quotient of total myocyte class volume and averagecell volume (Kajstura et al., 1995; Reiss et al., 1996). Number ofarteriole and capillary profiles per unit area of myocardium wasmeasured as previously done (Olivetti et al., 1991; Beltrami et al.,1994).

Sections were incubated with BrdU or Ki67 antibody. Myocytes (M) wererecognized with a mouse monoclonal anti-cardiac myosin, endothelialcells (EC) with a rabbit polyclonal anti-factor VIII and smooth musclecells (SMC) with a mouse monoclonal anti-α-smooth muscle actin myosin.The fractions of M, EC and SMC nuclei labeled by BrdU and Ki67 wereobtained by confocal microscopy (Orlic et al., 2001). Nuclei sampled in11 cytokine-treated mice; BrdU: M=3,541; EC=2,604; SMC=1,824. Ki67:M=3,096; EC=2,465; SMC=1,404.

BrdU was injected daily between days 14 to 26 to measure the cumulativeextent of cell proliferation while Ki67 was assayed to determine thenumber of cycling cells at sacrifice. Ki67 identifies cells in G1, S,G2, prophase and metaphase, decreasing in anaphase and telophase (Orlicet al., 2001). The percentages of BrdU and Ki67 positive myocytes were1.6- and 1.4-fold higher than EC, and 2.8- and 2.2-fold higher than SMC,respectively (FIG. 18C, 19). The forming myocardium occupied 76±11% ofthe infarct; myocytes constituted 61±12%, new vessels 12±5% and othercomponents 3±2%. The band contained 15×10⁶ regenerating myocytes thatwere in an active growing phase and had a wide size distribution (FIG.18D-E). EC and SMC growth resulted in the formation of 15±5 arteriolesand 348±82 capillaries per mm² of new myocardium. Thick wall arterioleswith several layers of SMC and luminal diameters of 10-30 μm representedvessels in early differentiation. At times, incomplete perfusion of thecoronary branches within the repairing myocardium during the fixationprocedure led to arterioles and capillaries containing erythrocytes(FIG. 18F-H). These results provided evidence that the new vessels werefunctionally competent and connected with the coronary circulation.Therefore, tissue repair reduced infarct size and myocyte growthexceeded angiogenesis; muscle mass replacement was the prevailingfeature of the infarcted heart.

E. Determination of Cell Differentiation

Cytoplasmic and nuclear markers were used. Myocyte nuclei: rabbitpolyclonal Csx/NRx2.5, MEF2, and GATA4 antibodies (Orlic et al., 2001;Lin et al., 1997; Kasahara et al., 1998); cytoplasm: mouse monoclonalnestin (Kachinsky et al., 1995), rabbit polyclonal desmin (Hermann andAebi, 1998), cardiac myosin, mouse monoclonal α-sarcomeric actin andrabbit polyclonal connexin 43 antibodies (Orlic et al., 2001). ECcytoplasm: mouse monoclonal flk-1, VE-cadherin and factor VIIIantibodies (Orlic et al., 2001; Yamaguchi et al., 1993; Breier et al.,1996). SMC cytoplasm: flk-1 and α-smooth muscle actin antibodies (Orlicet al., 2001; Couper et al., 1997). Scar was detected by a mixture ofcollagen type I and type III antibodies.

Five cytoplasmic proteins were identified to establish the state ofdifferentiation of myocytes (Orlic et al., 2001; Kachinsky et al., 1995;Hermann and Aebi, 1998): nestin, desmin, α-sarcomeric actin, cardiacmyosin and connexin 43. Nestin was recognized in individual cellsscattered across the forming band (FIG. 20A). With this exception, allother myocytes expressed desmin (FIG. 20B), α-sarcomeric actin, cardiacmyosin and connexin 43 (FIG. 20C). Three transcription factorsimplicated in the activation of the promoter of several cardiac musclestructural genes were examined (Orlic et al., 2001; Lin et al., 1997;Kasahara et al., 1998): Csx/NRx2.5, GATA-4 and MEF2 (FIG. 21A-C). Singlecells positive for flk-I and VE-cadherin (Yamaguchi et al., 1993; Breieret al., 1996), two EC markers, were present in the repairing tissue(FIG. 20D,E); flk-1 was detected in SMC isolated or within thearteriolar wall (FIG. 20F). This tyrosine kinase receptor promotesmigration of SMC during angiogenesis (Couper et al., 1997). Therefore,repair of the infarcted heart involved growth and differentiation of allcardiac cell populations resulting in de novo myocardium.

Example 3 Migration of Primitive Cardiac Cells in the Adult Mouse Heart

To determine whether a population of primitive cells was present in theadult ventricular myocardium and whether these cells possessed theability to migrate, three major growth factors were utilized aschemoattractants: hepatocyte growth factor (HGF), stem cell factor (SCF)and granulocyte monocyte colony stimulating factor (GM-CSF). SCF andGMCSF were selected because they have been shown to promotetranslocation of herriatopoietic stem cells. Although HGF inducesmigration of hematopoietic stem cells, this growth factor is largelyimplicated in mitosis, differentiation and migration of cardiac cellprecursors during early cardiogenesis. On this basis, enzymaticallydissociated cells from the mouse heart were separated according to theirsize. Methods for dissociating cardiac cells from heart tissue arewell-known to those skilled in the art and therefore would not involveundue experimentation (Cf U.S. Pat. No. 6,255,292 which is hereinincorporated by reference in its entirety) A homogenous population ofthe dissociated cardiac cells containing small undifferentiated cells,5-7 μm in diameter, with a high nucleus to cytoplasm ratio weresubjected to migration assay in Boyden microchambers characterized bygelatin-coated filters containing pores, 5 μm (Boyden et al., 1962, J.Exptl. Med. 115:453-456)

No major differences in the dose-response curve of migrated cells in thepresence of the three growth factors were detected. However, HGFappeared to mobilize a larger number of cells at a concentration of 100ng/ml. In addition, the cells that showed a chemotactic response to HGFconsisted of 15% of c-kit positive (c-kit^(POS)) cells, 50% of multidrugresistance-1 (MDR-1) labeled cells and 30% of stem cell antigen-1(Sca-1) expressing cells. When the mobilized cells were cultured in 15%fetal bovine serum, they differentiated into myocytes, endothelialcells, smooth muscle cells and fibroblasts. Cardiac myosin positivemyocytes constituted 50% of the preparation, while factor VIII labeledcells included 15%, alpha-smooth muscle actin stained cells 4%, andvimentin positive factor VIII negative fibroblasts 20%. The remainingcells were small undifferentiated and did not stain with these fourantibodies. In conclusion, the mouse heart possesses primitive cellswhich are mobilized by growth factors. HGF translocates cells that invitro differentiate into the four cardiac cell lineages.

Example 4 Cardiac C-Kit Positive Cells Proliferate In Vitro and GenerateNew Myocardium Vivo

To determine whether primitive c-kit^(POS) cells were present insenescent Fischer 344 rats, dissociated cardiac cells were exposed tomagnetic beads coated with c-kit receptor antibody (ACK-4-biotin,anti-c-kit mAb). Following separation, these small undifferentiatedcells were cultured in 10% fetal calf serum. Cells attached in a fewdays and began to proliferate at one week. Confluence was reached at7-10 days. Doubling time, established at passage P2 and P4, required 30and 40 hours, respectively. Cells grew up to P18 (90th generation)without reaching senescence. Replicative capacity was established byKi67 labeling: at P2, 88±14% of the cells contained Ki67 protein innuclei. Additional measurements were obtained between P1 and P4; 40% ofcells expressed alpha-sarcomeric actin or cardiac myosin, 13% desmin, 3%alpha-smooth muscle actin, 15% factor VIIII or CD31, and 18% nestin.Under these in vitro conditions, cells showed no clear myofibrillarorganization with properly aligned sarcomeres and spontaneouscontraction was never observed. Similarly, Ang II, norepinephrine,isoproterenol, mechanical stretch and electrical field stimulationfailed to initiate contractile function. On this basis, it was decidedto evaluate whether these cells pertaining to the myogenic, smoothmuscle cell and endothelial cell lineages had lost permanently theirbiological properties or their role could be reestablished in vivo.Following BrdU labeling of cells at P2, infarcted Fischer 344 rats wereinjected with these BrdU positive cells in the damaged region, 3-5 hoursafter coronary artery occlusion. Two weeks later, animals weresacrificed and the characteristics of the infarcted area were examined.Myocytes containing parallel arranged myofibrils along theirlongitudinal axis were recognized, in combination with BrdU labeling ofnuclei. Moreover, vascular structures comprising arterioles andcapillary profiles were present and were also positive to BrdU. Inconclusion, primitive c-kit positive cells reside in the senescent heartand maintain the ability to proliferate and differentiate intoparenchymal cells and coronary vessels when implanted into injuredfunctionally depressed myocardium.

Example 5 Cardiac Stem Cells Mediate Myocyte Replication in the Youngand Senescent Rat Heart

The heart is not a post-mitotic organ but contains a subpopulation ofmyocytes that physiologically undergo cell division to replace dyingcells. Myocyte multiplication is enhanced during pathologic overloads toexpand the muscle mass and maintain cardiac performance. However, theorigin of these replicating myocytes remains to be identified.Therefore, primitive cells with characteristics of stem/progenitor cellswere searched for in the myocardium of Fischer 344 rats. Young and oldanimals were studied to determine whether aging had an impact on thesize population of stem cells and dividing myocytes. The numbers ofc-kit and MDR1 positive cells in rats at 4 months were 11±3, and18±6/100 mm² of tissue, respectively. Values in rats at 27 months were35±10, and 42±13/100 mm². A number of newly generated small myocyteswere identified that were still c-kit or MDR1 positive. Ki67 protein,which is expressed in nuclei of cycling cells was detected in 1.3±0.3%and 4.1±1.5% of myocytes at 4 and 27 months, respectively. BrdUlocalization following 6 or 56 injections included 1.0±0.4% and 4.4±1.2%at 4 months, and 4.0±1.5% and 16±4% at 27 months. The mitotic indexmeasure tissue sections showed that the fraction of myocyte nuclei inmitosis comprised 82±28/10⁶ and 485±98/10⁶ at 4 and 27 months,respectively. These determinations were confirmed in dissociatedmyocytes to obtain a cellular mitotic index. By this approach, it waspossible to establish that all nuclei of multinucleated myocytes were inmitosis simultaneously. This information could not be obtained in tissuesections. The collected values showed that 95±31/10⁶ myocytes weredividing at 4 months and 620±98/10⁶ at 27 months. At both age intervals,the formation of the mitotic spindle, contractile ring, disassembly ofthe nuclear envelope, karyokinesis and cytokinesis were documented. Inconclusion, primitive undifferentiated cells reside in the adult heartand their increase with age is paralleled by an increase in the numberof myocytes entering the cell cycle and undergoing karyokinesis andcytokinesis. This relationship suggests that cardiac stem cells mayregulate the level and fate of myocyte growth in the aging heart.

Example 6 Chimerism of the Human Heart and the Role of Stem Cells

The critical role played by resident primitive cells in the remodelingof the injured heart is well appreciated when organ chimerism,associated with transplantation of a female heart in a male recipient,is considered. For this purpose, 8 female hearts implanted in male hostswere analyzed. Translocation of male cells to the grafted female heartwas identified by FISH for Y chromosome (see Example 1E). By thisapproach, the percentages of myocytes, coronary arterioles and capillaryprofiles labeled by Y chromosome were 9%, 14% and 7%, respectively.Concurrently, the numbers of undifferentiated c-kit and multidrugresistance-1 (MDR 1) positive cells in the implanted female hearts weremeasured. Additionally, the possibility that these cells contained the Ychromosome was established. Cardiac transplantation involves thepreservation of portions of the atria of the recipient on which thedonor heart with part of its own atria is attached. This surgicalprocedure is critical for understanding whether the atria from the hostand donor contained undifferentiated cells that may contribute to thecomplex remodeling process of the implanted heart. Quantitatively, thevalues of c-kit and, MDR1 labeled cells were very low in controlnon-transplanted hearts: 3 c-kit and 5 MDR1/100 mm² of left ventricularmyocardium. In contrast, the numbers of c-kit and MDR1 cells in theatria of the recipient were 15 and 42/100 mm². Corresponding values inthe atria of the donor were 15 and 52/100 mm² and in the ventricle 11and 21/100 mm². Transplantation was characterized by a marked increasein primitive undifferentiated cells in the heart. Stem cells in theatria of the host contained Y chromosome, while an average of 55% and63% of c-kit and MDR1 cells in the donor's atria and ventricle,respectively, expressed the Y chromosome. All c-kit and MDR1 positivecells were negative for CD45. These observations suggest that thetranslocation of male cells to the implanted heart has a major impact onthe restructuring of the donor myocardium. In conclusion, stem cells arewidely distributed in the adult heart and because of their plasticityand migration capacity generate myocytes, coronary arterioles andcapillary structures with high degree of differentiation.

Example 7 Identification and Localization of Stem Cells in the AdultMouse Heart

Turnover of myocytes occurs in the normal heart, and myocardial damageleads to activation of myocyte proliferation and vascular growth. Theseadaptations raise the possibility that multipotent primitive cells arepresent in the heart and are implicated in the physiological replacementof dying myocytes and in the cellular growth response following injury.On this basis, the presence of undifferentiated cells in the normalmouse heart was determined utilizing surface markers including c-kit,which is the receptor for stem cell factor, multidrug resistance-1(MDR1), which is a P-glycoprotein capable of extruding from the celldyes, toxic substances and drugs, and stem cell antigen-1 (Sca-1), whichis involved in cell signaling and cell adhesion. Four separate regionsconsisting of the left and right atria, and the base, mid-section andapical portion of the ventricle were analyzed. From the higher to thelower value, the number of c-kit positive cells was 26±11, 15±5, 10±7and 6±3/100 mm² in the atria, and apex, base and mid-section of theventricle, respectively. In comparison with the base and mid-section,the larger fraction of c-kit positive cells in the atria and apex wasstatistically significant. The number of MDR1 positive cells was higherthan those expressing c-kit, but followed a similar localizationpattern; 43±14, 29±16, 14±7 and 12±10/100 mm² in the atria, apex, baseand mid-section. Again the values in the atria and apex were greaterthan in the other two areas. Sca-1 labeled cells showed the highestvalue; 150±36/100 mm² positive cells were found in the atria. Cellspositive for c-kit, MDR1 and Sca-1 were negative for CD45, and formyocyte, endothelial cell, smooth muscle cell and fibroblast cytoplasmicproteins. Additionally, the number of cells positive to both c-kit andMDR1 was measured to recognize cells that possessed two stem cellmarkers. In the entire heart, 36% of c-kit labeled cells expressed MDR1and 19% of MDR1 cells had also c-kit. In conclusion, stem cells aredistributed throughout the mouse heart, but tend to accumulate in theregions at low stress, such as the atria and the apex.

Example 8 Repair of Infarcted Myocardium by Resident Cardiac Stem CellsMigration, Invasion and Expression Assays

The receptor of HGF, c-Met, has been identified on hematopoietic andhepatic stem cells (126, 90) and, most importantly, on satelliteskeletal muscle cells (92) and embryonic cardiomyocytes (127). Thesefindings prompted us to determine whether c-Met was present in CSCs andits ligand HGF had a biological effect on these undifferentiated cells.The hypothesis was made that HGF promotes migration and invasion of CSCsin vitro and favors their translocation from storage areas to sites ofinfarcted myocardium in vivo. HGF influences cell migration (128)through the expression and activation of matrix metalloproteinase-2 (94,95). This enzyme family may destroy barriers in the extracellular matrixfacilitating CSC movement, homing and tissue restoration.

IGF-1 is mitogenic, antiapoptotic and is necessary for neural stem cellmultiplication and differentiation (96, 97, 98). If CSCs express IGF-1R,IGF-1 may impact in a comparable manner on CSCs protecting theirviability during migration to the damaged myocardium. IGF-1overexpression is characterized by myocyte proliferation in the adultmouse heart (65) and this form of cell growth may depend on CSCactivation, differentiation and survival.

In the initial part of this study, migration and invasion assays wereconducted to establish the mobility properties of c-kit^(POS) andMDR1^(POS) cells in the presence of the chemotactic HGF.

Cardiac cells were enzymatically dissociated and myocytes were discarded(124). Small cells were resuspended in serum-free medium (SFM). Cellmigration was measured by using a modified Boyden chamber that had upperand lower wells (Neuro Probe, Gaithersburg, Md.). The filter for the48-well plate consisted of gelatin-coated polycarbonate membrane withpores of 5 μm in diameter. The bottom well was filled with SFMcontaining 0.1% BSA and HGF at increasing concentrations; 50 μl of smallcell suspension were placed in the upper well. Five hours later, filterswere fixed in 4% paraformaldehyde for 40 minutes and stained with PI,and c-kit and MDR1 antibodies. FITC-conjugated anti-IgG was used as asecondary antibody. Six separate experiments were done at each HGFconcentration. Forty randomly chosen fields were counted in each well ineach assay to generate a dose-response curve (FIG. 61). The motogeniceffects of IGF-1 on small cells was excluded by performing migrationassays with IGF-1 alone or in combination with HGF (data not shown).Invasion assays were done utilizing a chamber with 24-wells and 12 cellculture inserts (Chemicon, Temecula, Calif.). A thin layer of growthfactor-depleted extracellular matrix was spread on the surface of theinserts. Conversely, 100 ng/ml of HGF were placed in the lower chamber.Invading cells digested the coating and clung to the bottom of thepolycarbonate membrane. The number of translocated cells was measured 48hours later following the same protocol described in the migrationassay. Four separate experiments were done (FIG. 62). Consistent withthe results obtained in the migration assay, IGF-1 had no effects oncell invasion (data not shown).

Migration was similar in both cell types and reached its peak at 100ng/ml HGF. At 5 hours, the number of c-kit^(POS) and MDR1^(POS) cellstransmigrated into the lower chamber was 3-fold and 2-fold higher thancontrol cells, respectively. Larger HGF concentrations did not improvecell migration (FIGS. 61 and 62). On this basis, HGF at 100 ng/ml wasalso employed to determine the ability of c-kit^(POS) and MDR1^(POS)cells to penetrate the synthetic extracellular matrix of the invasionchamber. In 48 hours, the growth factor increased by 8-fold and 4-foldthe number of c-kit^(POS) and MDR1^(POS) cells in the lower portion ofthe chamber (FIGS. 61 and 62), respectively. IGF-1 had no effect on themobility of these CSCs at concentrations varying from 25 to 400 ng/ml.The addition of IGF-1 to HGF did not modify the migration and invasioncharacteristics of c-kit^(POS) and MDR1^(POS) cells obtained by HGFalone.

Small, undifferentiated c-Met^(POS) cells were collected withimmunomagnetic beads and the ability of these cells to cleave gelatinwas evaluated by zymography (FIG. 63). Briefly, small cells wereisolated from the heart (n=4) and subsequently separated by microbeads(Miltenyi, Auburn, Calif.) coated with c-Met antibody. Cells wereexposed to HGF, 100 ng/ml, for 30 minutes at 37° C. Cell lysates wererun onto 10% polyacrylamide gels copolymerized with 0.1% gelatin(Invitrogen, Carlsbad, Calif.). The gels were incubated in Coomassieblue staining solution (0.5%) and areas of gelatinolytic activity weredetected as clear bands against a gray background. This was done todemonstrate whether c-Met^(POS) cells expressed matrixmetalloproteinases (MMPs) and were capable of digesting the substratepresent in the gel (94, 95). Positive results were obtained (FIG. 63),suggesting that the mobility of these primitive cells was due, at leastin part, to activation of MMPs. Together, these in vitro assays point tothe chemotactic function of HGF on CSCs. Such a role of HGF appears tobe mediated by its binding to c-Met receptors and the subsequentstimulation of MMP synthesis (94, 95).

Myocardial Infarction in Mice

Myocardial infarction was produced in mice and 5 hours later 4 separateinjections of a solution containing HGF and IGF-1 were performed fromthe atria to the border zone. HGF was administrated at increasingconcentrations to create a chemotactic gradient between the stored CSCsand the dead tissue. This protocol was introduced to enhance homing ofCSCs to the injured area and to generate new myocardium. If this werethe case, large infarcts associated with animal death may be rapidlyreduced and the limits of infarct size and survival extended by thisintervention.

Female 129 SV-EV mice were used. Following anesthesia (150 mg ketamine-1mg acepromazine/kg b.w., i.m.), mice were ventilated, the heart wasexposed and the left coronary artery was ligated (61, 87). Coronaryligation in animals to be treated with growth factors was performed asclose as possible to the aortic origin to induce very large infarcts.Subsequently, the chest was closed and animals were allowed to recover.Five hours later, mice were anesthetized, the chest was reopened andfour injections of HGF-IGF-1, each of 2.5 μl, were made from the atriato the region bordering the infarct. The last two injections were doneat the opposite sides of the border zone. The concentration of HGF wasincreased progressively in the direction of the infarct, from 50 to 100and 200 ng/ml. IGF-1 was administered at a constant concentration of 200ng/ml. Mice were injected with BrdU (50 mg/kg b.w.) from day 6 to day 16to identify small, newly formed, proliferating myocytes during thisinterval. Sham-operated and infarcted-untreated mice were injected withnormal saline in the same four sites.

Before discussing the effects of CSCs on organ repair the presence ofc-Met and IGF-1R on cells expressing c-kit and MDR1 was measured in theatria and left ventricle (LV) of control mice. An identical analysis wasdone in the atria and infarcted and non-infarcted LV of mice subjectedto coronary artery occlusion. This determination was performed 2-3 hoursfollowing the administration of growth factors, which reflected 7-8hours after coronary occlusion (The objective was to document thatprimitive cells invaded the dead tissue and the surrounding viablemyocardium and that HGF and IGF-1 were implicated in this process.

c-Met and IGF-1R were detected in c-kit^(POS) and MDR1^(POS) cellsdispersed in regions of the normal (n=5), infarcted-treated (n=6) andinfarcted-untreated (n=5) heart (FIG. 22, A to F). A large fraction ofc-kit^(POS) and MDR1^(POS) cells expressed c-Met and IGF-1R alone or incombination. Myocardial infarction and the administration of growthfactors did not alter in a consistent manner the relative proportion ofCSCs with and without c-Met and IGF-1R in the myocardium (FIG. 64).Hairpin 1 (apoptosis) and hairpin 2 (necrosis) labeling and Ki67expression in nuclei (cycling cells) were used to establish theviability and activation of c-kit^(POS) and MDR1^(POS) cells in thevarious portions of the damaged and non-damaged heart, respectively(FIG. 22, G to L).

CSCs were more numerous in the atria than in the ventricle of controlmice. Acute myocardial infarction and growth factor administrationmarkedly changed the number and the distribution of primitive cells inthe heart. Viable c-kit^(POS) and MDR1^(POS) cells significantlyincreased in the spared myocardium of the border zone and remote tissueas well as in the dead myocardium of the infarcted region. Importantly,CSCs decreased in the atria (FIG. 22, M and N), suggesting that atranslocation of primitive cells occurred from this site of storage tothe stressed viable and dead myocardium. A different phenomenon wasnoted in infarcted-untreated mice, in which viable CSCs remained higherin the atria than in the ventricle. In control animals andinfarcted-treated mice, apoptosis and necrosis were not detected inc-kit^(POS) and MDR1^(POS) cells within the infarct and surroundingmyocardium. Ki67 labeling was identified in nearly 35% and 20% ofundifferentiated cells distributed in the border zone and in theinfarct, respectively (FIG. 65). In infarcted-untreated mice, themajority of c-kit^(POS) and MDR1^(POS) cells in the infarct wereapoptotic (FIG. 22, M and N). Necrosis was not seen. An apoptotic CSCdeath gradient was observed from the infarct to the distant myocardiumand atrial tissue. In these mice, only 10-14% of the viable c-kit^(POS)and MDR1^(POS) cells expressed Ki67 (FIG. 65).

Thus, these results support the notion that CSCs express c-Met andIGF-1R and, thereby, HGF and IGF-1 have a positive impact on thecolonization, proliferation and survival of CSCs in the infarcted heart.On the basis of in vitro and in vivo data, HGF appears to have aprevailing role in cell migration and IGF-1 in cell division andviability. In infarcted-untreated mice, however, CSCs do not translocateto the infarcted region and the pre-existing primitive cells die byapoptosis. The important question was then whether CSCs located withinthe infarct were capable of differentiating in the various cardiac celllineages and reconstitute dead myocardium. A positive finding wouldprovide a mechanism for cardiac repair in infarcted-treated mice and apotential explanation for the absence of myocardial regeneration ininfarcted-untreated mice.

For anatomical measurements, the heart was arrested in diastole withCdCl₂, and the myocardium was perfused with 10% formalin. The LV chamberwas filled with fixative at a pressure equal to the in vivo measuredend-diastolic pressure. The LV intracavitary axis was determined and themid-section was used to obtain LV thickness and chamber diameter.Infarct size was measured by the number of myocytes lost from the LVinclusive of the interventricular septum (87).

Myocardial infarction at 16 days resulted in a 42% (n=15) and 67% (n=22)loss of myocytes in the left ventricle and septum of untreated andHGF-IGF-1-treated mice, respectively (FIG. 23A). In spite of a 60%larger infarct, mice exposed to growth factors had a better preservationof cardiac function (FIG. 23B). HGF-IGF-1 led to a smaller elevation inLV end-diastolic pressure and a lesser decrease in +dP/dt and −dP/dt.The difference in infarct size did not influence mortality, which wassimilar in the two groups of mice: 43% in untreated and 40% in treated.Importantly, 14 of the 22 mice that received growth factors survivedwith infarcts affecting more than 60% of the LV. Seven of these mice hadinfarcts that involved 75% to 86% of LV. Untreated mice had infarctsthat never exceeded 60% (FIG. 23, C and D). In contrast to injectedmice, a portion of the posterior aspect of the LV wall and the entireinterventricular septum had to be preserved for untreated animals tosurvive. An infarct larger than 60% is incompatible with life in mice,rats, dogs and any other mammalian species. Irreversible cardiogenicshock and death supervene in humans with a 46% infarct (99).

From the volume of LV in sham-operated mice and infarct size inuntreated and treated animals it was possible to calculate the volume ofmyocardium destined to remain and destined to be lost 16 days aftercoronary artery occlusion. The volume of newly formed myocardiuminclusive of myocytes, vascular structures and other tissue componentswas detected exclusively in growth factor-treated mice and found to be 8mm³. Thus, the repair band reduced infarct size from 67% to 57% (FIGS.68 and 69).

The chemotactic and mitogenic properties of HGF-IGF-1 resulted in themobilization, proliferation and differentiation of primitive cells inthe infarcted region of the wall creating new myocardium. In spite ofthe complexity of this methodological approach in small animals, theformation of a myocardial band within the infarct was obtained in 85% ofthe cases (22 of 26 mice). The band occupied 65±8% of the damaged areaand was located in the mid-portion of the infarct equally distant fromthe inner and outer layer of the wall. In very large infarcts, theentire thickness of the wall was replaced by developing myocardium (FIG.23, E to H).

Anatomically, the longitudinal axis and the chamber diameter weresimilar in the two groups of infarcted mice indicating that thetherapeutic intervention promoted positive ventricular remodeling. Thisnotion was consistent with the 60% larger infarct size in treated mice.Additionally, the wall thickness-to-chamber radius ratio decreased lessin treated than in untreated mice. This relationship, in combinationwith the smaller increase in LV end-diastolic pressure in treated micesignificantly attenuated the increase of diastolic wall stress in thisgroup (FIG. 67).

Primitive cells were labeled with monoclonal c-kit and MDR1 antibodies(82, 83). BrdU incorporation was detected by BrdU antibody (61, 87).Endothelial cells were recognized with anti-factor VIII and smoothmuscle cells with anti-α-smooth muscle actin. For myocytedifferentiation, nestin, desmin, cardiac myosin, α-sarcomeric actin,N-cadherin and connexin 43 antibodies were utilized. Scar formation inthe infarct was detected by a mixture of anti-collagen type I and typeIII (83, 61, 87).).

The composition of the repairing myocardium was evaluatedmorphometrically. Antibodies specific for myocytes, endothelial cellsand smooth muscle cells were employed for the recognition of parenchymalcells and vessel profiles (61, 87). Moreover, BrdU labeling of cells wasused as a marker of regenerating tissue over time. Myocytes occupied84±3% of the band, the coronary vasculature 12±3%, and other structuralcomponents 4±1%. New myocytes varied from 600 to 7,200 μm³, with anaverage volume of 2,200±400 μm³ (FIGS. 68 and 69). Together, 3.1±1.1million myocytes were formed to compensate for a loss of 2.4±0.8 millioncells. This slight excess in cell regeneration was at variance withmyocyte size. In sham-operated hearts, myocyte volume, 18,000±3,600 μm³,was 8.2-fold larger than growing cells. Importantly, 16% of the musclemass lost was reconstituted 16 days after infarction (lost muscle mass:18,000×2.4×10⁶=43 mm³; regenerated muscle mass: 2,200×3.1×10⁶=7.0 mm³;7.0:43=16%). The new myocytes were still maturing, but functionallycompetent as demonstrated echocardiographically in vivo and mechanicallyin vitro.

Echocardiography was performed in conscious mice by using an AcusonSequoia 256c equipped with a 13-MHz linear transducer (87).Two-dimensional images and M-mode tracings were recorded from theparastemal short axis view at the level of papillary muscles. Ejectionfraction (EF) was derived from LV cross-sectional area in 2D short axisview: EF=[(LVDA−LVSA)/LVDA]×100, where LVDA and LVSA correspond to LVareas in diastole and systole. For hemodynamics, mice were anesthetizedand a Millar microtip pressure transducer connected to a chart recorderwas advanced into the LV for the evaluation of pressures and + and−dP/dt in the closed-chest preparation. Echocardiography performed atday 15 showed that contractile activity was partially restored in theregenerating portion of the wall of treated infarcts. Ejection fractionwas also higher in treated than in untreated mice (FIG. 24, A to E).Thus, structural repair was coupled with functional repair.

To confirm that new myocytes reached functional competence andcontributed to the amelioration of ventricular performance, these cellswere enzymatically dissociated from the regenerating myocardium of theinfarcted region of the wall (129) and their contractile behavior wasevaluated in vitro (124, 130). Myocytes isolated from infarcted treatedmice (n=10) by collagenase digestion were placed in a cell bath (30±0.2°C.) containing 1.0 mM Ca²⁺ and stimulated at 0.5 Hz by rectangulardepolarizing pulses, 3-5 ms in duration in twice diastolic threshold inintensity. Parameters were obtained from video images stored in acomputer (124, 130). Developing myocytes were small with myofibrilslocated at the periphery of the cell in the subsarcolemma) region. Thenew myocytes resembled neonatal cells actively replicating DNA. Theywere markedly smaller than the spared hypertrophied ventricular myocytes(FIG. 25, A and B). In comparison with surviving old myocytes, growingcells showed a higher peak shortening and velocity of shortening, and alower time to peak shortening (FIG. 25, C to J).

The isolated newly generated myocytes were stained by Ki67 to determinewhether these cells were cycling and, therefore, synthesizing DNA. Anidentical protocol was applied to the isolated surviving hypertrophiedmyocytes of infarcted-treated mice. On this basis, the DNA content ofeach myocyte nucleus in mononucleated and binucleated cells wasevaluated by PI staining and confocal microscopy (see FIG. 25, A and B).Control diploid mouse lymphocytes were used as baseline. The objectivewas to establish if cell fusion occurred in CSCs before their commitmentto cell lineages. This possibility has recently been suggested by invitro studies (131, 132). Non-cycling new myocytes and enlarged sparedmyocytes had only diploid nuclei, excluding that such a phenomenonplayed a role in cardiac repair (FIG. 66).

To establish the level of differentiation of maturing myocytes withinthe band, the expression of nestin, desmin, cardiac myosin heavy chain,α-sarcomeric actin, N-cadherin and connexin 43 was evaluated. N-cadherinidentifies the fascia adherens and connexin 43 the gap junctions in theintercalated discs. These proteins are developmentally regulated.Connexin 43 is also critical for electrical coupling and synchrony ofcontraction of myocytes. These 6 proteins were detected in essentiallyall newly formed myocytes (FIG. 26, A to N). The percentage of myocyteslabeled by BrdU was 84±9%, indicating that cell proliferation wasongoing in the regenerating tissue. Cardiac repair included theformation of capillaries and arterioles (FIG. 27, A to D). The presenceof red blood cells within the lumen indicated that the vessels wereconnected with the coronary circulation. This phase of myocardialrestoration, however, was characterized by a prevailing growth ofresistance arterioles than capillary structures. There were 59±29arterioles and 137±80 capillaries per mm² of new myocardium.

The current findings indicate that resident CSCs can be mobilized fromtheir region of storage to colonize the infarcted myocardium where theydifferentiate into cardiac cell lineages resulting in tissueregeneration. The intervention utilized here was capable of salvaginganimals with infarct size normally incompatible with life in mammals.

Example 9 Cardiac Stem Cells Differentiate In Vitro Acquiring FunctionalCompetence In Vivo

A. Collection and Cloning of Cells

Cardiac cells were isolated from female Fischer rats at 20-25 months ofage (111, 112). Intact cells were separated and myocytes were discarded.Small cells were resuspended and aggregates removed with a strainer.Cells were incubated with a rabbit c-kit antibody (H-300, Santa Cruz)which recognizes the N-terminal epitope localized at the external aspectof the membrane (121). Cells were exposed to magnetic beads coated withanti-rabbit IgG (Dynal) and c-kit^(POS) cells were collected with amagnet (n=13). For FACS (n=4), cells were stained withr-phycoerythrin-conjugated rat monoclonal anti-c-kit (Pharmingen). Withboth methods, c-kit^(ms) cells varied from 6-9% of the small cellpopulation.

c-kit^(POS) cells scored negative for myocyte (α-sarcomeric actin,cardiac myosin, desmin, α-cardiac actinin, connexin 43), endothelialcell (EC; factor VIII, CD31, vimentin), smooth muscle cell (SMC;α-smooth muscle actin, desmin) and fibroblast (F; vimentin) cytoplasmicproteins. Nuclear markers of myocyte lineage (NRx2.5, MEF2, GATA-4) weredetected in 7-10% and cytoplasmic proteins in 1-2% of the cells.c-kit^(POS) cells did not express skeletal muscle transcription factors(MyoD, myogenin, Myf5) or markers of the myeloid, lymphoid and erythroidcell lineages (CD45, CD45RO, CD8, TER-119), indicating the cells wereLin⁻ c-kit^(POS) cells.

c-kit^(POS) cells were plated at 1−2×10⁴ cells/ml NSCM utilized forselection and growth of neural stem cells (122). This was composed byDulbecco's MEM and Ham's F12 (ratio 1:1), bFGF, 10 ng/ml, EGF, 20 ng/ml,HEPES, 5 mM, insulin-transferrin-selenite. c-kit^(POS) cells attached intwo weeks and began to proliferate (FIG. 28 a, b). NSCM was thensubstituted with differentiating medium (DM) and confluence was reachedin 7-10 days. Cells were passaged by trypsinization. Cycling cells, asdetermined by Ki67 expression, varied from 74±12% to 84±8% at passages(P) P1-P5 (n=5 at each P). Doubling time at P2 and P4 averaged 41 hours.Cells continued to divide up to P23 without reaching growth arrest andsenescence, at which time cells were frozen. Cardiac lineages wereidentified from P0 to P23. At P0 (n=7), P3 (n=10), P10 (n=13) and P23(n=13), myocytes were 29-40%, EC 20-26%, SMC 18-23% and F 9-16%.Aliquots of P23 grown after 6 months in liquid nitrogen expressed thesame phenotypes as the parental cells.

At P0 and P1 when grown in DM, 50% of the cells exhibited NRx2.5, 60%MEF2, 30% GATA-4 and 55% GATA-5 (FIG. 28 c-f). Conversely, skeletalmuscle (MyoD, myogenin, Myf5), blood cell (CD45, CD45RO, CD8, TER-119)and neural (MAP1b, neurofilament 200, GFAP) markers were not identified.

For cloning, cells were seeded at 10-50 cells/ml NSCM (FIG. 28 g) (109,110). After one week, colonies derived from a single cell wererecognized (FIG. 28 h); fibronectin, procollagen type I and vimentinwere absent excluding the fibroblast lineage. Individual colonies weredetached with cloning cylinders and plated. Multiple clones developedand one clone in each preparation was chosen for characterization. MEMcontaining 10% FCS and 10⁻⁸ M dexamethasone was employed to inducedifferentiation (DM). For subcloning, cells from multiple clones wereplated at 10-50 cells/ml NSCM. Single subclones were isolated and platedin DM. At each subcloning step, an aliquot of cells was grown insuspension to develop clonal spheres.

Each clone contained groups of 2-3 Lin⁻ c-kit^(POS) cells (FIG. 29 a),although the majority of these cells (˜20-50) were dispersed amongc-kit^(NEG) cells. Some cells were Ki67 positive and occasionally inmitosis (FIG. 29 b-d). Myocytes expressing cardiac myosin andα-sarcomeric actin, EC expressing factor VIII, CD31 and vimentin, SMCexpressing α-smooth muscle actin and F expressing vimentin alone wereidentified in each clone (FIG. 29 e-h). Aggregates of small cellscontaining nestin were also present (Supplementary Information). Thus,Lin⁻ c-kit^(POS) cells isolated from the myocardium possessed theproperties expected for stem cells. They were clonogenic, self-renewingand multipotent and gave origin to the main cardiac cell types.Subclonal analysis of several primary clones confirmed the stability ofthe phenotype of the primary clones: clonogenicity, self-renewal andmultipotentiality. The phenotype of most subclones was indistinguishablefrom that of the primary clones. However, in two of eight subclones,only myocytes were obtained in one case and exclusively EC wereidentified in the other.

Clonogenic cells, grown in suspension in Coming untreated dishesgenerated spherical clones (FIG. 30 a). This anchorage independentgrowth is typical of stem cells^(14,15). Spheroids consisted of clustersof c-kit^(POS) and c-kit^(NEG) cells and large amounts of nestin (FIG.30 b-a). Similarly to other stem cells^(14,15), following plating in DM,spheroids readily attached, and cells migrated out of the spheres anddifferentiated (FIG. 30 e-h).

Cells were fixed in 4% paraformaldehyde and undifferentiated cells werelabeled with c-kit antibody. Markers for myocytes included NRx2.5, MEF2,GATA-4, GATA-5, nestin, α-sarcomeric actin, α-cardiac actinin, desminand cardiac myosin heavy chain. Markers for SMC comprised α-smoothmuscle actin and desmin, for EC factor VIII, CD31 and vimentin, and forF vimentin in the absence of factor VIII, fibronectin and procollagentype I. MyoD, myogenin and Myf5 were utilized as markers of skeletalmuscle cells. CD45, CD45RO, CD8 and TER-119 were employed to excludehematopoietic cell lineages. MAP1b, neurofilament 200 and GFAP were usedto recognize neural cell lineages. BrdU and Ki67 were employed toidentify cycling cells (61, 87). Nuclei were stained by PI.

Myocytes and SMC failed to contract in vitro. Angiotensin II,isoproterenol, norepinephrine and electrical stimulation did not promotecontraction. EC did not express markers of full differentiation such aseNOS.

B. Myocardial Infarction and Cell Implantation

BrdU labeled cells (P2; positive cells=88±6%) were implanted. Myocardialinfarction was produced in female Fischer rats at 2 months of age (111).Five hours later, 22 rats were injected with 2×10⁵ cells in two oppositeregions bordering the infarct; 12 rats were sacrificed at 10 days and 10rats at 20 days. At each interval, 8-9 infarcted and 10 sham-operatedrats were injected with saline and 5 with Lin⁻ c-kit^(NEG) cells andused as controls. Under ketamine anesthesia, echocardiography wasperformed at 9 and 19 days, only in rats killed at 20 days. From M-modetracings, LV end-diastolic diameter and wall thickness were obtained.Ejection fraction was computed (87). At 10 and 20 days, animals wereanesthetized and LV pressures and + and −dP/dt were evaluated in theclosed-chest preparation (111). Mortality was lower but notstatistically significant in treated than in untreated rats at 10 and 20days after surgery, averaging 35% in all groups combined. Protocols wereapproved by the institutional review board.

C. Anatomic and Functional Results

Hearts were arrested in diastole and fixed with formalin. Infarct sizewas determined by the fraction of myocytes lost from the left ventricle(87), 53±7% and 49±10% (NS) in treated and untreated rats at 10 days,and 70±9% and 55±10% (P<0.001) in treated and untreated rats at 20 days,respectively. The volume of 400 new myocytes was measured in each heart.Sections were stained with desmin and laminin and PI. In longitudinallyoriented myocytes with centrally located nuclei, cell length anddiameter across the nucleus were collected to compute cell volume (87).

Sections were incubated with BrdU and Ki67 antibodies. A band ofregenerating myocardium was identified in 9 of 12 treated infarcts at 10days, and in all 10 treated infarcts at 20 days. At 10 days, the bandwas thin and discontinuous and, at 20 days, was thicker and presentthroughout the infarcted area (FIG. 31 a-c). Myocytes (M), EC, SMC and Fwere identified by cardiac myosin, factor VIII, α-smooth muscle actinand vimentin in the absence of factor VIII, respectively. Myocytes werealso identified by cardiac myosin antibody and propidium iodide (PI). At10 and 20 days, 30 and 48 mm³ of new myocardium were measured,respectively. Tissue regeneration reduced infarct size from 53±7% to40±5% (P<0.001) at 10 days, and from 70±9% to 48±7% (P<0.001) at 20days.

Cells labeled by BrdU and Ki67 were identified by confocal microscopy(103, 105). The number of nuclei sampled for BrdU labeling were:M=5,229; EC=3,572; SMC=4,010; F=5,529. Corresponding values for Ki67were: M=9,290; EC=9,103; SMC=8,392. Myocyte differentiation wasestablished with cardiac myosin, α-sarcomeric actin, α-cardiac actinin,N-cadherin and connexin 43. Collagen was detected by collagen type I andtype III antibodies.

Since implanted cells were labeled by BrdU, the origin of the cells inthe developing myocardium was identified by this marker. Myocytes,arterioles (FIG. 31 f-n) and capillary profiles were detected. At 10days, the proportion of myocytes, capillaries and arterioles was lower,and collagen was higher than at 20 days. Cell growth evaluated by Ki67was greater at 10 days decreasing at 20 days (SupplementaryInformation).

Cardiac myosin, α-sarcomeric actin, α-cardiac actinin, N-cadherin andconnexin 43 were detected in myocytes (FIG. 31 o-p; SupplementaryInformation). At 10 days, myocytes were small, sacrcomeres were rarelydetectable and N-cadherin and connexin 43 were mostly located in thecytoplasm (FIG. 31 o). Myocyte volume averaged 1,500 μm³ and 13.9×10⁶myocytes were formed. At 20 days, myocytes were closely packed andmyofibrils were more abundant; N-cadherin and connexin 43 defined thefascia adherens and nexuses in intercalated discs (FIG. 31 p). Myocytevolume averaged 3,400 μm³ and 13×10⁶ myocytes were present.

Myocyte apoptosis was measured by in situ ligation of hairpinoligonucleotide probe with single base overhang. The number of nucleisampled for apoptosis was 30,464 at 10 days and 12,760 at 20 days. Thepreservation of myocyte number from 10 to 20 days was consistent with adecrease in Ki67 labeling and an increase in apoptosis (0.33±0.23% to0.85±0.31%, P<0.001).

Thus, myocyte proliferation prevailed early and myocyte hypertrophylater. From 10-20 days, the number of vessels nearly doubled.

Procedures for determining mechanical properties of the new myocyteshave been previously described³⁰. Myocytes isolated from infarctedtreated rats (n=4) were placed in a cell bath (30±0.2° C.) containing1.0 mM Ca²⁺ and stimulated at 0.5 Hz by rectangular depolarizing pulses,3-5 ms in duration in twice diastolic threshold in intensity. Mechanicalparameters were obtained from video images stored in a computer. Themechanical behavior of myocytes isolated from the infarcted andnon-infarcted regions of treated hearts was measured at 20 days (FIG. 32a-e). New cells were calcium tolerant and responded to stimulation.However, in comparison with spared myocytes, maturing cells showed adecreased peak shortening and velocity of shortening; time to peakshortening and time to 50% re-lengthening were similar in the two groupsof cells (FIG. 33 a-h). Developing myocytes had myofibrils mostlydistributed at the periphery; sarcomere striation was apparent (FIG. 32a-e).

Cell implantation reduced infarct size and cavitary dilation, andincreased wall thickness and ejection fraction. Contraction reappearedin the infarcted ventricular wall and end-diastolic pressure, developedpressure and + and −dP/dt improved at 20 days. Diastolic stress was 52%lower in treated rats (Supplementary Information). Thus, structural andfunctional modifications promoted by cardiac repair decreased diastolicload and ameliorated ventricular performance. This beneficial effectoccurred in spite of the fact that infarct size was similar in the twogroups of rats.

Colonization, replication, differentiation of the transplanted cells andtissue regeneration required c-kit^(POS) cells and damaged myocardium.c-kit^(POS) cells injected in sham-operated rats grafted poorly and didnot differentiate. Injection of c-kit^(NEG) cells in the border ofinfarcts had no effect on cardiac repair.

The multipotent phenotype of the Lin⁻ c-kit^(POS) cell reported here isin apparent contrast with cardiac cell lineage determinations in chicken(113), zebrafish (114) and mammals (115) concluding that myocytes, SMC,and EC each originates from a separate lineage. However, not all studiesare in agreement (116). Because these experiments (113, 114, 115, 116)did not address the developmental potential of any of the cells marked,as has been done here, the different outcomes likely represent anotherexample of the difference between normal developmental fate anddevelopmental potential. Additionally, the plasticity of human embryonicstem cells (117), progenitor endothelial cells (101) and clonogeniccells (52) as means to repair damaged myocardium has recently beendocumented (101, 52).

Example 10 Mobilization of Cardiac Stem Cells (CSC) by Growth FactorsPromotes Repair of Infarcted Myocardium Improving Regional and GlobalCardiac Function in Conscious Dogs

The methods of the previous non-limiting examples were used withexceptions as described below.

Myocardial regeneration after infarction in rodents by stem cell homingand differentiation has left unanswered the question whether a similartype of cardiac repair would occur in large mammals. Moreover, whethernew myocardium can affect the functional abnormality of infarctedsegments restoring contraction is not known. For this purpose, dogs werechronically instrumented for measurements of hemodynamics and regionalwall function. Stroke volume and EF were also determined. Myocardialinfarction was induced by inflating a hydraulic occluder around the leftanterior descending coronary artery. Four hours later, HGF and IGF-1were injected in the border zone to mobilize and activate stem cells;dogs were then monitored up to 30 days. Growth factors induced chroniccardiac repair reversing bulging of the infarct: segment shorteningincreased from −2.0±0.7% to +5.5±2.2%, stroke work from −18±11 to +53±10mm×mmHg, stroke volume from 22±2 to 45±4 ml and ejection fraction from39±3 to 64±4%. In treated dogs at 8 hours after infarction, the numberof primitive cells increased from 240±40 c-kit positive cells atbaseline to 1700±400 (remote myocardium), 4400±1200 (border zone) and3100±900 c-kit positive cells/100 mm² (infarcted area). Ki67 labelingwas detected in 48%, 46% and 26% of c-kit positive cells in the remote,border and infarcted myocardium, respectively. Thus, high levels ofthese cells were replicating. These effects were essentially absent ininfarcted untreated dogs. Acute experiments were complemented with thequantitative analysis of the infarcted myocardium defined by theimplanted crystals 10-30 days after coronary occlusion. Changes fromparadoxical movement to regular contraction in the new myocardium werecharacterized by the production of myocytes, varying in size from 400 to16,000 with a mean volume of 2,000±640 μm³. Resistance vessels withBrdU-labeled endothelial and smooth muscle cells were 87±48 per mm² oftissue. Capillaries were 2-3-fold higher than arterioles. Together,16±9% of the infarct was replaced by healthy myocardium. Thus, canineresident primitive cells can be mobilized from the site of storage toreach dead myocardium. Stem cell activation and differentiation promotesrepair of the infarcted heart improving local wall motion and systemichemodynamics.

Example 11 Mobilization of Resident Cardiac Stem Cells Constitutes anImportant Additional Treatment to Angiotensin II Blockade in theInfarcted Heart

The methods of the previous non-limiting examples were used withexceptions as described below.

Two of the major complicating factors of myocardial infarction (MI) arethe loss of muscle mass and cavitary dilation, which both contribute tonegative left ventricular (LV) remodeling and to the depression incardiac performance. In an attempt to interfere with these deleteriouseffects of MI, resident cardiac stem cells (CSC) were mobilized andactivated to promote tissue regeneration, and the AT₁, receptor blockerlosartan (Los) was administered, 20 mg/kg body weight/day, to attenuatecellular hypertrophy, and, thereby, the expansion in chamber volume. Onthis basis, MI was produced in mice and the animals were subdivided infour groups: 1. Sham-operated (SO); 2. MI only; 3. MI-Los; 4.MI-Los-CSC. One month after MI, animals were sacrificed, and LVfunction, infarct dimension and cardiac remodeling were evaluated.Myocardial regeneration was also measured in mice treated with CSC.Infarct size, based on the number of myocytes lost by the LV was 47% inMI, 51% MI-Los and 53% MI-Los-CSC. In comparison with MI and MI-Los, MItreated with Los and CSC resulted in a more favorable outcome of thedamaged heart in terms of chamber diameter: −17% vs MI and −12% vsMI-Los; longitudinal axis: −26% (p<0.001) vs MI and −8% (p<0.02) vsMI-Los; and chamber volume: −40% (p<0.01) vs MI and −35% (p<0.04) vsMI-Los. The LV-mass-to-chamber volume ratio was 47% (p<0.01) and 56%(p<0.01) higher in MI-Los-CSC than in MI and MI-Los, respectively.Tissue repair in MI-Los-CSC was made of 10×10⁶ new myocytes of 900 μm³.Moreover, there were 70 arterioles and 200 capillaries per mm² ofmyocardium in this group of mice. The production of 9 mm³ of newmyocardium reduced MI size by 22% from 53% to 41% of LV.Echocardiographically, contractile function reappeared in the infarctedregion of the wall of mice with MI-Los-CSC. Hemodynamically, MI-Los-CSCmice had a lower LVEDP, and higher + and −dP/dt. In conclusion, thepositive impact of losartan on ventricular remodeling is enhanced by theprocess of cardiac repair mediated by translocation of CSC to theinfarcted area. Mobilized CSC reduce infarct size and ventriculardilation and, thereby, ameliorate further the contractile behavior ofthe infarcted heart.

Example 12 Hepatocyte Growth Factor (HGF) Induces the Translocation ofc-met to the Nucleus Activating the Expression of GATA-4 and CardiacStem Cell (CSC) Differentiation

The methods of the previous non-limiting examples were used withexceptions as described below.

In preliminary studies we were able to document that CSCs positive forc-kit or MDR-1 expressed the surface receptor c-met. c-met is thereceptor of HGF and ligand binding promoted cell motility via thesynthesis of matrix metalloproteinases. However, it was unknown whetherc-met activation had additional effects on CSCs biology and function.For this purpose, we tested whether c-met on CSCs exposed to 50 ng/ml ofHGF in NSCM responded to the growth factor by internalization andtranslocation within the cell. Surprisingly, a localization of c-met inthe nucleus was detected by confocal microscopy in these stimulatedcells which maintained primitive characteristics. This unusual impact ofHGF on c-met raised the possibility that the mobilized receptor couldinteract with other nuclear proteins participating in cell growth anddifferentiation of CSCs. Because of the critical role of the cardiacspecific transcription factor GATA-4 in the commitment of cell lineage.By immunoprecipitation and Western blot, a protein complex made by c-metand GATA-4 was identified. A time-dependent analysis following a singleHGF stimulation showed a progressive increase in c-met-GATA-4 complexfrom 15 minutes to 3 days. Time was also coupled with differentiation ofprimitive cells into myocytes and other cardiac cells. To establish amolecular interaction at the DNA level between GATA-4 and c-met, a gelretardation assay was performed on nuclear extracts isolated from cellsstimulated with HGF for 1 hour. A shifted band was obtained utilizing aprobe containing the GATA sequence. However, the addition of GATA-4antibody resulted in a supershifted band. Conversely, the inclusion ofc-met antibody attenuated the optical density of the GATA band. Since aGATA sequence upstream to the TATA box was identified in the c-metpromoter, a second mobility shift assay was performed. In this case,nuclear extracts from HGF stimulated cells resulted in a shifted bandwhich was diminished by c-met antibody. In contrast, GATA-4 antibodyinduced a supershifted band. Thus, HGF-mediated translocation of c-metat the level of the nucleus may confer to c-met a function oftranscription factor and future studies will demonstrate whether thisDNA binding enhances the expression of GATA-4 leading to thedifferentiation of immature cardiac cells.

Example 13 Isolation and Expansion of Human Cardiac Stem Cells andPreparation of Media Useful Therein

Myocardial tissue (averaging 1 g or less in weight) was harvested understerile conditions in the operating room.

Growth media was prepared using 425-450 ml of DMEM/F12 (Cambrex12-719F), 5-10% patient serum (50-75 ml of serum derived from 100-150 mlof patient's blood, obtained along with the atrial appendage tissue), 20ng/ml human recombinant bFGF (Peprotech 100-18B), 20 ng/ml humanrecombinant EGF (Sigma E9644), 5 μg/ml insulin (RayBiotech IP-01-270), 5μg/ml transferrin (RayBiotech IP-03-363), 5 ng/ml sodium selenite (SigmaS5261), 1.22 mg/ml uridine (Sigma U-3003) and 1.34 mg/ml inosine (Sigma1-1024).

The tissue was immersed inside a sterile Petri dish filled with growthmedium, and then cut under sterile conditions into small pieces (200-400mg). Each tissue piece was then transferred into 1.2 ml cryogenic vialscontaining 1 ml of freezing medium (the freezing medium is composed ofthe growth culture medium mixed with DMSO in a 9:1 volumetric mixture;e.g., 9 ml of medium mixed with 1 ml of DMSO).

The cryogenic vials were frozen in a nalgene container pre-cooled at−70° C. to −80° C. and then stored at −70 to −80° C. for at least 3days.

Samples were thawed (at 37° C.) via immersion in a container containing70% ethanol in distilled water placed in a water bath warmed to 37° C.After 2 minutes, the vial was taken under the hood and opened, and thesupernatant was removed by pipetting and substituted with normal salinesolution kept at room temperature. The sample was then transferred to a100 mm Petri dish and washed twice with saline solution. Forcepssterilized in Steri 250 (Inotech) were used to manually separatefibrotic tissue and fat from the cardiac specimen. Samples were thentransferred to the growth medium and minced in 1-2 mm² slices.

Slices were plated in uncoated dishes under a cover slide containinggrowth medium enriched with 5-10% human serum as described above. Petridishes were placed in an incubator at 37° C., under 5% CO₂.

One-two weeks after tissue seeding, outgrowth of CSCs was apparent. Thegrowth medium was changed twice a week for the entire period of cellexpansion. The medium was stored at 4° C. and was warmed at 37° C. priorto use. A total of 8 ml of medium was used in a 100 mm Petri dish. In anattempt to preserve the conditioned medium created by the culturedpieces or cells, only 6 ml of medium were removed and 6 ml of freshmedium were added at a time.

After an additional two weeks, a cluster of ˜5,000 myocardial cells wasexpected to surround each tissue fragment.

At subconfluence, the growth medium was removed and cells were detachedwith 4 ml of trypsin (0.25%) [Carnbrex cat #10170; negligible level ofendotoxin] per dish for 5-7 minutes. The reaction was stopped with 6 mlof medium containing serum.

Cells were then sorted to obtain c-kit^(POS) cells using Myltenyiimmunomagnetic beads. Cell sorting was performed through the indirecttechnique utilizing anti-c-kit H-300 (sc-5535 Santa Cruz) as the primaryantibody and anti-rabbit conjugated with microbeads as the secondaryantibody (130048602 Miltenyi). Cells outgrown from the myocardial samplewere placed in 15 ml Falcon tubes and centrifuged at 850 g at 4° C. for10 minutes. The medium was discarded and cells were re-suspended in 10ml PBS. Cells were centrifuged again at 850 g at 4° C. for 10 minutes asa wash. The PBS was removed and the pellet of cells re-suspended in 975μl PBS before being transferred to a 1.5 ml tube. 25 μl of anti-c-kitantibody (corresponding to 25 .mu.g of antibody) H-300 (sc-5535 SantaCruz) was added. The incubation with the antibody was carried out at 4°C. for one hour with the vials in a shaker having 360 degree rotation.

Following incubation, cells were centrifuged at 850 g at 4° C. for 10minutes and resuspended in 1 ml PBS and centrifuged again. Cells werethen incubated with the secondary antibody conjugated with immunobeads(80 μl PBS and 20 μl antibody) for 45 minutes at 4° C. with the vials ina shaker having 180 degree rotation. After the incubation, 400 μl of PBSwere added and the cell suspension was passed through a separationcolumn for magnetic sorting (Miltenyi 130042201). The c-kit positivecells attached to the column and were recovered and placed in a 1.5 mltube. Cells were centrifuged and resuspended in 1 ml of pre-warmed (37°C.) medium and plated in 24-well plates.

c-kit+ cells were then plated in growth medium for expansion. After 3-4months (±1 month), approximately 1 million cells was obtained. Thegrowth medium was changed twice a week for the entire period of cellexpansion. The medium was stored at 4° C. and was again warmed at 37° C.prior to use. A total of 1 ml of medium was used in each of the 24wells. To obtain the desired number of cells to be injected, cells werepassaged three times at subconfluence: 1) 35-mm Petri dishes filled with2 ml of medium; 2) 60-mm Petri dishes filled with 4 ml of medium; and 3)100-mm Petri dishes filled with 8 ml of medium. To preserve theconditioned medium created by the cultured cells, only ⅔ of the mediumwas changed at each passage.

The characteristics of c-kit+ cells (CSCs) were analyzed byimmunocytochemistry and FACS using antibodies against c-kit and againstmarkers of cardiac lineage commitment (i.e., cardiac myocytes,endothelial cells, smooth muscle cells), which include: (a)transcription factors such as GATA4, MEF2C, Ets1, and GATA6 and (b)other antigens such as α-sarcomeric actin, troponin I, MHC, connexin 43,N-cadherin, von Willebrand factor and smooth muscle actin. If desired,it is also possible to analyze the cells for other markers and/orepitopes including flik-1.

To activate the CSCs, the CSCs were incubated for two hours with growthmedia additionally containing 200 ng/ml hepatocyte growth factor and 200ng/ml insulin-like growth factor-1.

Example 14 Isolation and Expansion of Human Cardiac Stem Cells and Usein Treatment of Myocardial Infarction

Discarded myocardial specimens were obtained from 51 consenting patientswho underwent cardiac surgery as described above. Samples were mincedand seeded onto the surface of uncoated Petri dishes containing a mediumsupplemented with hepatocyte growth factor and insulin-like growthfactor-I at concentrations of 200 ng/ml and 200 ng/ml, respectively.Successful outgrowth of cells was obtained in 29 cases. In this subset,outgrowth of cells was apparent at ˜4 days after seeding and, at ˜2weeks, clusters of ˜5,000-7,000 cells surrounded each tissue fragment(FIG. 70A-C). Cells outgrown from the tissue were sorted for c-kit withimmunobeads and cultured (Beltrami, 2003; Linke, 2005). Cell phenotypewas defined by FACS and immunocytochemistry as described above(Beltrami, 2003; Orlic, 2001; Urbanek, 2005). Sorted-c-kit^(POS)-cellswere fixed and tested for markers of cardiac, skeletal muscle, neuraland hematopoietic cell lineages (Table 1, below) to detect lineagenegative (Lin⁻)-hCSCs (Beltrami, 2003; Linke, 2005; Urbanek, 2005). Afraction of cells outgrown from the myocardial samples at P0 expressedthe stem cell antigens c-kit, MDR1 and Sca-1-like (FIG. 70D-F); theyconstituted 1.8±1.7, 0.5±0.7, and 1.3±1.9 percent of the entire cellpopulation, respectively. These cells were negative for hematopoieticcell markers including CD133, CD34, CD45, CD45RO, CD8, CD20, andglycophorin A (Table 1, below).

TABLE 1 Identification of Lineage Negative (Lin−) CSCs and EarlyCommitted cells (ECCs) Markers Lin−CSCs ECCs Labeling Hematopoieticlineage GATA1^(§) absent absent Direct GATA2^(§) absent absent DirectCD45* absent absent Direct CD45RO* absent absent Direct CD8* absentabsent Direct CD20* absent absent Direct Glycophorin A* absent absentDirect Skeletal muscle lineage Myo D^(§) absent absent DirectMyogenin^(§) absent absent Direct Myf5^(§) absent absent Direct Skeletalmyosin^(†) absent absent Direct Neural lineage Neurofilament 200^(†)absent absent Direct GFAP^(§) absent absent Indirect MAP1b^(§) absentabsent Indirect Myocyte lineage GATA4^(§) absent present DirectNkx2.5^(‡) absent present Direct MEF2C^(‡) absent present Direct Cardiacmyosin^(†) absent present Indirect/QD α-sarcomeric actin absent presentIndirect/QD Nestin absent present Indirect Desmin absent presentIndirect Connexin 43 absent present Indirect/QD N-Cadherin absentpresent Indirect/QD Vascular smooth muscle lineage GATA4^(§) absentpresent Direct GATA6^(‡) absent present Direct α-smooth muscle actin^(†)absent present Indirect/QD TGFβ1 receptor absent present IndirectEndothelial cell lineage GATA4^(§) absent present Direct Ets1^(‡) absentpresent Direct Erg1^(‡) absent present Direct Vimentin absent presentIndirect Von Willebrand Factor^(†) absent present Indirect/QDVE-Cadherin absent present Indirect Flk1 absent present Indirect Table1: Direct labeling technique corresponds to the utilization of afluorochrome-conjugated primary antibody while indirect labelingtechnique requires the use of non-conjugated primary antibody andfluorochrome-conjugated secondary antibody. Mixtures offluorochrome-conjugated primary antibodies were utilized: *Cocktail 1,^(§)Cocktail 2, ^(†)Cocktail 3 and ^(‡)Cocktail 4. QD indicates directlabeling of primary antibodies with quantum dots (QD); indirect/QDindicates that both indirect labeling and direct labeling with QD wereemployed.

The cardiac transcription factor GATA4 and the myocyte transcriptionfactor MEF2C were present in some of these cells. A large fraction ofcells expressed myocyte, SMCs, and EC cytoplasmic proteins. Some cellswere positive for neurofilament 200 (FIG. 70G-J). FACS analysis ofunfractionated cells confirmed the data obtained by immunolabeling. Forcytochemistry, when possible, antibodies were directly labeled byfluorochromes or quantum dots to avoid cross-reactivity andautofluorescence (Table 1, online) (Linke, 2005; Urbanek, 2005).Antibodies employed for FACS of unfractionated cells and c-kit^(POS)cells are listed in Table 2 (Beltrami, 2003; Urbanek, 2005).

TABLE 2 Antibodies for FACS Analysis Antibody Company Labeling TechniqueCD8 (T lymphocytes) BD Pharmingen Direct (FITC) CD 20 (B lymphocytes) BDPharmingen Direct (PECy5) CD31 (PECAM-1) eBioscience Direct (PE) CD34(Sialomucin) Miltenyi Direct (FITC) CD45 (Pan-leukocyte marker) MiltenyiDirect (FITC) CD45RO (T lymphocytes) Santa Cruz Indirect CD71(Transferrin receptor) BD Pharmingen Direct (PE) CD117 (c-kit) SantaCruz Indirect CD133 (prominin-like 1) Miltenyi Direct (PE) CD243 (MDR)BD Pharmingen Direct (FITC) Glycopohrin A (erythrocytes) BD PharmingenDirect (FITC) flk1 (VEGFR-2) AbCam Indirect Table 2: Direct labelingtechnique corresponds to the utilization of a fluorochrome-conjugatedprimary antibody while indirect labeling technique requires the use of anon-conjugated primary antibody and a fluorochrome-conjugated secondaryantibody (PE: phycoerythrin; FITC: fluoroisithiocyanate). MDR: multidrugresistance; PECAM-1: platelet endothelial cell adhesion molecule 1;VEGF-R2: vascular endothelial growth factor receptor 2.

Because of previous results in animals (Beltrami, 2003; Linke, 2005),cells were sorted for c-kit at P0 with immunobeads. Thec-kit^(POS)-cells included Lin⁻ cells, 52±12 percent, and earlycommitted cells, 48±12 percent (FIG. 71A). C-kit^(POS)-cells plated inthe presence of human serum attached rapidly and continued to grow up toP8, undergoing ˜25 population doublings. Cells maintained a stablephenotype and did not reach growth arrest or senescence at P8. Thepercentage of e-kit^(POS)-cells did not vary from P1 to P8, averaging71±8 percent. Ki67^(POS)-cycling cells (FIG. 71B) remained constant fromP1 to P8, averaging 48±10 percent. However, 6±4 percent of cellsexpressed p16^(INK4a), a marker of cellular senescence (FIG. 71C). FACSanalysis showed that c-kit^(POS)-cells continued to be negative forhematopoietic cell lineages and a large fraction expressed thetransferrin receptor CD71, which correlates closely with Ki67 (FIG.71D). The undifferentiated state of c-kit^(POS)-cells, 63±6 percent, wasestablished by the absence of nuclear and cytoplasmic proteins ofcardiac cells (Table 1). The intermediate filament nestin, which isindicative of sternness, was found in 62±14 percent of thec-kit^(POS)-cells (FIG. 71E-G).

Cloning Assay

Human c-kit^(POS)-cells were sorted at P0 and, under microscopiccontrol, individual c-kit^(POS) cells were seeded in single wells ofTerasaki plates at a density of 0.25-0.5 cells/well (FIG. 71H)(Beltrami, 2003; Linke, 2005). Wells containing more than one cell wereexcluded; 50±10 percent of the c-kit^(POS)-cells to be deposited wereLin⁻. BrdU (10 μM) was added 3 times a day for 5 days (Beltrami, 2003;Linke, 2005). After ˜3-4 weeks, 53 small clones were generated from6,700 single seeded cells. Thus, c-kit^(POS)-hCSCs had 0.8 percentcloning efficiency. The number of cells in the clones varied from 200 to1,000 (FIG. 71I). Of the 53 clones, 12 did not grow further. Theremaining 41 clones were expanded and characterized byimmunocytochemistry. Doubling time was 29±10 hours and 90±7 percent ofcells after 5 days were BrdU^(POS). Dexamethasone was employed to inducedifferentiation (Beltrami, 2003; Linke, 2005), and as a result, cardiaccell lineages were detected. They included myocytes, SMCs, and ECs (FIG.71J). Myocytes were the predominant cell population and were followed byECs and SMCs (FIG. 75).

Myocardial Infarction

Myocardial infarction was produced in anesthetized femaleimmunodeficient Scid mice (Urbanek, 2005) and Fischer 344 rats(Beltrami, 2003) treated with a standard immunosuppressive regimen(Zimmermann, 2002). C-kit^(POS)-cells were isolated and expanded frommyocardial samples of 8 patients (˜3 specimens/patient) who underwentcardiac surgery as described above. In these studies, c-kit^(POS)-cellswere collected at P2 when 200,000 c-kit^(POS)-cells were obtained fromeach sample. This protocol required ˜7 weeks. Shortly after coronaryocclusion, two injections of ˜40,000 human-c-kit^(POS)-cells were madeat the opposite sites of the border zone (Beltrami, 2003; Orlic, 2001;Lanza, 2004). Animals were exposed to BrdU and sacrificed 2-3 weeksafter infarction and cell implantation (Beltrami, 2003; Orlic, 2001;Urbanek, 2005; Lanza, 2004). Echocardiography was performed 2-3 daysbefore measurements of left ventricular (LV) pressures and dP/dt(Beltrami, 2003; Orlic, 2001; Urbanek, 2005; Lanza, 2004). The heart wasarrested in diastole and fixed by perfusion with formalin. In eachheart, infarct size and the formation of human myocytes, arterioles, andcapillaries was determined (Anversa, 2002).

Repair was obtained in 17 of 25 treated-mice (68 percent), and 14 of 19treated-rats (74 percent). To interpret properly the failure toreconstitute infarcts, c-kit^(POS)-cells were injected together withrhodamine-labeled microspheres for the recognition of the sites ofinjection and correct administration of cells (Leri, 2005; Kajstura,2005). The unsuccessfully treated-animals were considered an appropriatecontrol for the successfully treated-animals. For completeness, 12immunodeficient infarcted mice and 9 immunosuppressed infarcted ratswere injected with PBS and used as additional controls. Infarct size wassimilar in all groups averaging 48±9 percent in mice and 52±12 percentin rats.

Human myocardium was present in all cases in which humanc-kit^(POS)-cells were delivered properly within the border zone ofinfarcted mice and rats. These foci of human myocardium were locatedwithin the infarct and were recognized by the detection of human DNAsequences with an Alu probe (Just, 2003). The extent of reconstitutionof the lost myocardium was 1.3±0.9 mm³ in mice and 3.7±2.9 mm³ in rats(FIG. 72A-C). The accumulation of newly formed cells was also determinedby BrdU labeling of structures; BrdU was given to the animals throughoutthe period of observation. Although human c-kit^(POS)-cells wereobtained from 8 patients, there were no apparent differences in terms ofdegree of cardiac repair with the various human cells. The variabilityin tissue regeneration was independent from the source of the cells,suggesting that other factors affected the recovery of the treatedheart.

The formation of human myocardium was confirmed by the recognition ofhuman Alu DNA sequence in the infarcted portion of the wall of treatedrats. Additionally, human MLC2v DNA sequence was identified togetherwith the human Alu DNA (FIG. 72D). The surviving myocardium in the sameanimals did not contain human Alu or human MLC2v DNA sequences. Theviable myocardium showed rat MLC2v DNA.

In treated-mice, human myocardium consisted of closely packed myocytes,which occupied 84±6 percent of the new tissue while resistancearterioles and capillary profiles together accounted for 7±3 percent.Corresponding values in treated-rats were 83±8 and 8±4 percent.Dispersed human myocytes, SMCs and ECs together with isolated humanvascular profiles were detected, scattered throughout the infarct (FIG.76). Human myocytes, SMCs and ECs were not found in unsuccessfullyinjected infarcted mice and rats or in animals treated with PBS.

In Situ Hybridization and PCR

Human cells were detected by in situ hybridization with FITC-labeledprobe against the human-specific Alu repeat sequences (Biogenex) (Just,2003). Additionally, human X-chromosomes, and mouse and ratX-chromosomes were identified (Quaini, 2002). DNA was extracted fromtissue sections of the viable and infarcted LV of rats treated withhuman cells. PCR was conducted for human Alu (approximately 300 basepairs in length; specifically found in primate genomes; is present inmore than 10% of the human genome; and is located with an averagedistance of 4 kb in humans), and rat and human myosin light chain 2vsequences (See Table 3 below).

TABLE 3  Recognition of Rat and Human Cells: Detection  of Myosin Light Chain 2v Gene and Alu Sequence.Rat myosin light chain 2v primers: rMyl2-S: CCTCTAGTGGCTCTACTGTAGGCTTC(SEQ ID NO: 1; 26mer, melting temperature 55° C.)rMyl2-A: TTCCACTTACTTCCACTCCGAGTCC(SEQ ID NO: 2; 25mer, melting temperature 59° C.)Human myosin light chain 2v primers: hMLC2-S: GACGTGACTGGCAACTTGGACTAC(SEQ ID NO: 3; 24mer, melting temperature 57° C.)hMLC2-A: TGTCGTGACCAAATACACGACCTC(SEQ ID NO: 4; 24mer, melting temperature 58° C.) Alu sequence primer:ARC-261r: GAGACGGAGTCTCGCTCTGTCGC(SEQ ID NO: 5; 23mer, melting temperature 61° C.) Each sample was mixedwith 15 μl Platinum PCR BlueMix solution (Invitrogen) and 0.2 μM of eachprimer and subject to PCR. The PCR reaction was performed as follows:94° C. for 30 sec; 35 cycles of 94° C. for 30 sec, 60° C. for 30 sec,and 72° C. for 1 min; 72° C. for 3 min. PCR products were separated on2% agarose gel electrophoresis.

To avoid unspecific labeling with secondary antibodies, most primaryantibodies were directly labeled by fluorochromes (Table 1). In spite ofthis precaution, it is impossible by this approach to eliminate minimallevels of autofluorescence inherent in tissue sections (Leri, 2005;Linke, 2005; Urbanek, 2005). To exclude this source of artifact, whenpossible, primary antibodies were conjugated with quantum dots; theexcitation and emission wavelength of these semiconductor particles isoutside the range of autofluorescence, eliminating this confoundingvariable (Leri, 2005). Quantum dot labeling was applied to theidentification of transcription factors, cytoplasmic and membraneproteins of cardiomyocytes, SMCs and ECs within the band of regeneratedhuman myocardium.

Following the recognition of human cells by the Alu probe, cardiacmyosin heavy chain and troponin I were detected in new myocytes togetherwith the transcription factors GATA4 and MEF2C. Additionally, thejunctional proteins connexin 43 and N-cadherin were identified at thesurface of these developing myocytes (FIG. 72E-J). Laminin was alsoapparent in the interstitium. Human myocytes varied significantly insize from 100 to 2,900 μm³ in both animal models (FIG. 77).

Female human cells were injected in female infarcted mice and rats.Therefore, human X-chromosomes were identified together with mouse andrat X-chromosomes to detect fusion of human cells with mouse or ratcells. No colocalization of human X-chromosome with a mouse or ratX-chromosome was found in newly formed myocytes, coronary arterioles,and capillary profiles (FIG. 73H-M). Importantly, human myocytes, SMCsand ECs carried at most two X-chromosomes. Therefore, cell fusion didnot play a significant role in the formation of human myocardium in thechimeric infarcted hearts.

Characteristics of the Human Myocardium

Vasculogenesis mediated by the injection of human c-kit^(POS)-cells wasdocumented by coronary arterioles and capillaries constitutedexclusively by human SMCs and ECs (FIG. 73A-F). There was no visibleintegration of human SMCs and ECs in mouse or rat coronary vasculature.In no case, we found vessels formed by human and non-human cells. Thenumber of human arterioles and capillaries was comparable in rats andmice and there was one capillary per 8 myocytes in both cases (FIG.73G). Additionally, the diffusion distance for oxygen averaged 18 μm.These capillary parameters are similar to those found in the late fetaland newborn human heart (Anversa, 2002).

The Human Myocardium is Functionally Competent

To determine whether the regenerated human myocardium was functionallycompetent and restored partly the function of the infarcted heart,echocardiograms were examined retrospectively following the histologicaldocumentation of transmural infarcts and the presence or absence ofnewly formed human myocardium (FIG. 74A-C; FIG. 78). Myocardialregeneration was associated with detectable contractile function in theinfarcted region of the wall; this was never the case in the absence oftissue reconstitution. The formation of human myocardium increased theejection fraction of the infarcted ventricle (FIG. 74D). Moreover,myocardial regeneration attenuated chamber dilation, increasedLV-mass-to-chamber volume ratio (FIG. 74E), and improved globalventricular function by limiting the elevation in LVEDP and the decreasein LVDP and positive and negative dP/dt after infarction (FIG. 74F).

Where relevant, results provided throughout this example are mean±SD.Significance was determined by Student's t test and Bonferroni method(Anversa, 2002).

Example 15 Formation of Large Coronary Arteries by Cardiac Stem Cells—aBiological Bypass

To compare the effect of Injection of clonogenic EGFPPOS-c-kitPOS-CSCs(non-activated-CSCs) and EGFPPOS-c-kitPOS-CSCs, activated with HGF andIGF-1 (activated-CSCs) on vasculature occlusion, the left coronaryartery of Fischer 344 rats was occluded following standard procedures.Non-activated CSCs or activated-CSCs (activation occurred 2 hours priorto their implantation) were implanted in proximity to the occluded leftcoronary artery. Because of the anatomical location of the ligature, thesites of cell implantation were away from the infarcted region of theventricular wall that resulted from the ligature (FIG. 82).Non-activated-CSCs seeded within the myocardium showed a high apoptoticrate that increased progressively from 12 and 24 to 48 hours afterdelivery (FIG. 83). Cell death led to a complete disappearance of theimplanted cells in a period of 1-2 weeks. Conversely, striking positiveeffects were detected with implantation of CSCs activated by growthfactors (FIG. 79 a). The activated CSCs homed to the myocardium where,acutely, apoptosis prevailed on cell replication and, subsequently, celldivision exceeded cell death (FIG. 79 b-d).

Although activated- and non-activated-CSCs accumulated within thenon-damaged myocardium at the sites of injection, cell engraftment wasrestricted to activated-cells. Engraftment requires the synthesis ofsurface proteins that establish cell-to-cell contact and the interactionbetween cells and extracellular matrix (Lapidot, 2005). Connexin 43, N-and E-cadherin, and L-selectin were expressed only in a large fractionof activated-CSCs (FIG. 79E-G). These junctional and adhesion proteinswere absent in the clusters of non-activated-CSCs in the myocardium.

Apoptosis never affected engrafted cells and involved exclusivelynon-engrafted cells (FIG. 79H). This phenomenon was consistent withanoikis of the non-engrafted cells, wherein programmed cell deathtriggered by lack of cell-to-cell contacts (Frisch, 2001; Melendez,2004).

To verify that activation of CSCs by growth factors played a role incell engraftment, and that cell engraftment was independent fromischemic damage, activated-CSCs were injected in the intact myocardiumof control non-infarcted rats. One month later, a large quantity ofcells was present in the epicardial region of the heart (FIG. 79I).These cells expressed connexin 43 and 45, N- and E-cadherin andL-selectin. The implanted cells preserved their undifferentiatedphenotype, most likely due to the absence of tissue damage and thenecessity to regenerate lost myocardium (Beltrami, 2003; Orlic, 2001;Mouquet, 2005).

Quantitative measurements at 2 days after treatment showed that only ˜5%(4,800±2,600) of the 80,000-100,000 injected non-activated-CSCs werepresent in the myocardium. Following the delivery of activated-CSCs,large quantities of cells expressing EGFP were detected. However, theywere clearly less than the total number of administered cells,48,000±13,000. These cells were the product of death and division of thenon-engrafted and engrafted CSCs, respectively.

To determine whether the changes in myocardial environment created bycoronary occlusion influenced the differentiation of activated-CSCs intovascular smooth muscle (SMCs) and endothelial cells (ECs), theexpression of hypoxia-inducible factor-1 (HIF-1), which is atranscriptional regulator of the SDF-1 chemokine12, and SDF-1 wasdetermined as both are upregulated with ischemia (Abott, 2004; Ceradini,2005) and may correlate with the oxygen gradient within the tissue(Butler, 2005). This myocardial response was seen in longitudinalsections of the infarcted heart in which hypoxia increased progressivelyfrom the base to the mid-portion and apex of the infarcted ventricle.Conversely, the expression of HIF-1 and SDF-1 was minimal in the deadmyocardium of the apex, modest in the mid-region, and highly apparenttowards the ischemic but viable myocardium of the base. HIF-1 and SDF-1were restricted to the endothelial lining of the vessel wall.Immunolabeling was consistent with the regional expression of HIF-1 andSDF-1 by Western blotting and the levels of SDF-1 measured by ELISA.

The effects of activated-CSCs on the development of conductive coronaryarteries and their branches in the infarcted heart were evaluated at 2weeks and one month after coronary ligation and treatment. These timepoints were selected because maturation of the coronary tree postnatallyin the rat requires approximately one month (Anversa, 2002), although asignificant magnitude of vessel growth occurs within 10-15 days afterbirth (Olivetti, 1980; Rakusan, 1984). At 2 weeks after infarction andcell implantation, newly formed large EGFP-positive coronary arterieswere found in the epimyocardium in close proximity to the site ofinjection (FIG. 80 a, b). The generated vessels permeated the survivingmyocardium and the border of the infarct at the base of the heart nearthe occluded coronary artery. Conductive arteries with diameter ≧150 μmpossessed an internal elastic lamina and were restricted to the viablemyocardium of the base and upper mid-region of the ventricle. Forcomparison, the origin of the left coronary artery has a diameter of˜275 μm. No newly formed EGFP-positive myocytes were found in thesurviving myocardium adjacent to or distant from the regeneratedvessels. This provides evidence of a selective response of CSCs to theregional needs of the organ, which appear to condition stem cell growthand differentiation (Baxter, 2000).

The presence of small resistance arterioles with a diameter <25 μm werelimited to the scarred infarcted area (FIG. 80 c). Resistance arteriolesof this size were not detected within the spared myocardium at 2 weeks.Similarly, a small number of capillaries were present but only in theinfarcted myocardium. In all cases, the vessel wall was composedexclusively of EGFP-positive SMCs and ECs. There were no EGFP-negativeSMCs or ECs in the regenerated vessels. This excludes the possibility ofa cooperative role of existing SMCs and ECs and lineage commitment ofactivated-CSCs in vessel formation. Vasculogenesis appeared to be theonly mechanism of vessel growth under these conditions.

Observations were taken one-month after infarction and cell-therapy todetermine whether the formed coronary vasculature represented temporaryvessels that subsequently atrophied, or functionally competent vessels,which grew further with time. This interval was relevant not only forthe detection of additional vascular growth but also for thecharacterization of infarct healing. Infarct healing is completed in ˜4weeks in rodents (Fishbein, 1978) and leads to the accumulation ofcollagen type III and type I within the necrotic tissue. The scarredmyocardium contains, at most, a few scattered vascular profiles sincethe vessels present early during healing progressively die by apoptosis(Cleutjens, 1999). Therefore, the distribution of different classes ofEGFP-positive coronary vessels was measured in the infarcted andnon-infarcted myocardium at 2 weeks and one month.

Numerous EGFP-positive coronary vessels with diameters ranging from 6 to250 μm were present at one month in both the viable myocardium andinfarcted region of the wall suggesting that time resulted in anexpansion of the coronary vasculature. At one month, large,intermediate, and small-sized coronary arteries and arterioles togetherwith capillary profiles were detected in both the spared myocardium andinfarcted portion of the ventricular wall. As noted at 2 weeks, theregenerated vessels were composed only by EGFP-positive SMCs and ECs(FIG. 84). These observations were supported by quantitative results,which indicated that all classes of coronary vessels had developed atone month (FIG. 80 d). Thus, activated CSCs are capable of generating denovo the various segments of the rat coronary vascular tree.

To assess the actual growth potential of CSCs, whether thereconstitution of coronary artery classes and capillary profilesinvolved fusion events (Wagers, 2004) between resident ECs and SMCs andinjected CSCs was determined. The formation of heterokaryons wasestablished by measuring sex-chromosomes in the nuclei of EGFP-positiveECs and SMCs within the vessel wall (Urbanek, 2005a; Urbanek, 2005b;Dawn, 2005). Since female clonogenic CSCs were implanted in femalehearts, the number of X-chromosomes in newly formed vessels wasidentified by FISH (FIG. 80 e). In all cases, at most two X-chromosomeswere found in regenerated ECs and SMCs, suggesting that cell fusion, ifpresent, played a minor role in the restoration of the coronaryvasculature by activated-CSCs.

To determine whether the new epicardial coronary vessels werefunctionally connected with the aorta and the existing coronarycirculation, an ex vivo preparation was employed. The heart wascontinuously perfused retrogradely through the aorta with an oxygenatedTyrode solution containing rhodamine-labeled dextran (MW 70,000; redfluorescence). This molecule does not cross the endothelial barrier, andit allows the visualization of the entire coronary vasculature bytwo-photon microscopy (Urbanek, 2005; Dawn, 2005). Due to the scatteringof laser light by biological structures (Helmchen, 2005), this analysiswas restricted to the outermost ˜150 μm of the epimyocardium; theventricular wall has a thickness of ˜2.0 mm. Resident and generatedcoronary vessels were distinguished by the absence and presence of EGFPlabeling (green-fluorescence) of the wall, respectively. Tissue collagenwas detected by second harmonic generation (blue fluorescence), which isthe result of two-photon excitation and the periodic structure ofcollagen (Schenke-Layland, 2005). A discrete localization of collagenwas assumed to correspond to viable myocardium while extensiveaccumulation of collagen was interpreted as representative of infarctedmyocardium.

Perfusion from the aorta with dextran identified large vessels, nearly200 μm in diameter and EGFP-positive-wall, within the non-infarctedepimyocardium of treated rats at 2 weeks (FIG. 81 a). Minimal amounts ofcollagen were seen in proximity of the vessel wall. Similar vessels werealso found in the scarred myocardium at 2 weeks and one month (FIG. 81b-e). At times, the new coronary vessels traversed the epicardial regionof the infarct (FIG. 81 e), which was partly replaced by EGFP-positivecells, corresponding to discrete foci of myocardial regeneration (notshown). When resolution permitted, a direct connection betweenpre-existing (EGFP-negative-wall) and generated (EGFP-positive-wall)coronary vessels was recognized (FIG. 81 f), documenting integration ofthese temporally distinct, old and new, segments of the coronaryvascular tree.

The improvement in coronary circulation with cell treatment wasassociated with attenuation of ventricular dilation and relativeincreases in wall thickness-to-chamber radius ratio and ventricularmass-to-chamber volume ratio (FIG. 81 g). These anatomical variableshave dramatic impacts on ventricular function and myocardial loading(Pfeffer, 1990). As expected, regeneration of the coronary circulationdid not decrease infarct size (FIG. 81 g). Cell therapy was introducedshortly after coronary ligation and the cardiomyocytes supplied by theoccluded coronary artery were dead by 4-6 hours (Anversa, 2002).However, the hemodynamic alterations in left ventricular end-diastolicpressure, developed pressure, positive and negative dP/dt, and diastolicstress were all partly reduced by the amelioration of coronary perfusionmediated by cell therapy (FIG. 81 h).

Example 16 Catheter-Based Intracoronary Delivery of Cardiac Stem Cellsin a Large Animal Model

15 pigs underwent thoracotomy for the dual purpose of: 1) resecting andharvesting atrial appendage tissue, and 2) inducing myocardialinfarction through occlusion of the distal left anterior descendingcoronary artery for a 90 min period, followed by reperfusion. CSCs wereharvested from atrial appendages, cultured and expanded ex vivo asdescribed above, and then injected intracoronarily in the same pig 2-3months later (average, 86 days). 7 pigs received intracoronary CSCsinjections while 8 pigs received vehicle injections. All pigs underwentserial testing for cardiac markers, 2D echocardiographic examinations,and (in a subset) invasive hemodynarnic monitoring as well as detailedhistopathological examination of their internal organs. There was noevidence of untoward effects related to the CSC treatment in the heartor in the various organs examined histopathologically, and treated pigsdemonstrated a trend towards improved cardiac function. These resultsconfirmed the safety and feasibility of intracoronary delivery of CSCsin this large animal model of ischemic cardiomyopathy.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

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The invention claimed is:
 1. A pharmaceutical composition comprisingisolated adult cardiac stem cells in a therapeutically effective amountfor repairing and/or regenerating damaged myocardium and apharmaceutically acceptable carrier, wherein said adult cardiac stemcells are c-kit positive, lineage negative, and characterized by theirability to differentiate into myocytes, endothelial cells, and smoothmuscle cells.
 2. The pharmaceutical composition of claim 1, wherein thecardiac stem cells are isolated from myocardial tissue.
 3. Thepharmaceutical composition of claim 1, wherein the cardiac stem cellsare human cells.
 4. The pharmaceutical composition of claim 3, whereinthe cardiac stem cells are autologous.
 5. The pharmaceutical compositionof claim 1, wherein the cardiac stem cells express c-Met and/or IGF-1receptor.
 6. The pharmaceutical composition of claim 1, wherein thecardiac stem cells are 5-7 μm in diameter.
 7. The pharmaceuticalcomposition of claim 1, wherein the composition comprises about 2×10⁴ toabout 1×10⁵ isolated adult cardiac stem cells.
 8. The pharmaceuticalcomposition of claim 1, wherein the cardiac stem cells are cultured andexpanded in vitro.
 9. The pharmaceutical composition of claim 1, furthercomprising one or more cytokines.
 10. The pharmaceutical composition ofclaim 9, wherein the one or more cytokines are hepatocyte growth factorand/or insulin-like growth factor-1.
 11. The pharmaceutical compositionof claim 1, wherein the composition is formulated for intramyocardial orintraarterial injection.
 12. The pharmaceutical composition of claim 1,wherein the cardiac stem cells do not express lineage markers MyoD,myogenin, Myf5, CD45, CD45RO, CD8, TER-119, MAP1b, neurofilament 200,GFAP, α-sarcomeric actin, cardiac myosin, desmin, α-cardiac actinin,factor VIII, CD31, vimentin, or α-smooth muscle actin.